Suportado pelo NIH R01 HL74082, a Fundação Hartwell, ea Fundação Cirurgia Focused Ultrasound.

1. Produção de microbolhas <ol><li> Para preparar microbolhas de albumina (MBs), coloque uma solução a 1% de albumina de soro em solução salina em um frasco com um cobertor de gás (octafluoropropane) acima da fase aquosa. Brevemente sonicate a solução (30 seg) com um desintegrador de ultra-som equipado com um longo ½ &quot;sonda de titânio. Esta formulação é semelhante ao Optison (GE Heathcare), que é fornecido em uma faixa de concentração de 0,5-1,2 x 10 9 MBs / ml. Determine significa MB diâmetro com um Coulter Multisizer Contador. O diâmetro MB albumina média utilizada neste estudo foi 1.93um ± 1.63um. </li><li> Para fabricar MBs lipídico, preparar uma dispersão aquosa de 1 mg / ml estearato de polietilenoglicol-40 (Sigma Chemical Co., St. Louis, MO) e 2 mg / ml fosfatidilcolina distearoyl (Lipídeos Avanti Polar, Alabaster, AL) e sonciate como anteriormente descrito (1.1) com um gás decafluorobutane. Determine o diâmetro médio MB com um contador Coulter Multisizer. O diâmetro médio de lipídios MB utilizado neste estudo foi 2.01um ± 1.29um. </li></ol> 2. Fabricação de nanopartículas <ol><li> Estes métodos foram adaptados a partir da água-em-óleo-em-água técnica de evaporação de emulsão de solvente descrito por Davda (2002) e Chappell (2008). </li><li> Prepare a 2% de poli (álcool vinílico) PVA solução dissolvendo 20g de PVA em 1000 ml de água deionizada (DI). Deixa-se dissolver completamente em uma placa de agitação durante a noite. Solução centrífuga a 1000 rpm por 10 minutos e filtrar com um filtro de 0.22μm estéril para remover qualquer PVA residual não dissolvido. </li><li> Para fabricar albumina sérica bovina (BSA) carregado poli (láctico-co-ácido glicólico) (Plaga) nanopartículas (NPs), dissolver 180mg de estreantes 85:15 Plaga em 6ml cloreto de metileno (MC) em um frasco de cintilação de vidro. Vortex solução MC / Plaga por 2 minutos. </li><li> Dissolver carga desejada (15mg de BSA) em 1,5 ml de PBS. Adicionar a solução de PBS / BSA em duas partes para a solução Plaga / MC com vórtex intermitente. Solução de colocar no gelo por 5 minutos e sonicate em 45W por 120 segundos. </li><li> Adicione o MC / Plaga / carga / solução PBS para 24ml de 2% PVA, em duas parcelas com vórtex intermediária. </li><li> Colocar a solução em gelo por 5 minutos e sonicate em 45W para 120. </li><li> Agitar a emulsão NP por 12 horas em um prato misture um exaustor para permitir a evaporação do MC e estabilização NP. </li><li> Centrifugar a suspensão por 25 minutos a 20.000 rpm a 4 ° C por duas vezes para isolar e remover NPs PVA residual. Ressuspender o sedimento em 8ml de água DI e centrifugar por 10 minutos a 1000 rpm a 4 ° C para isolar a população 100nm de NPs. </li><li> Isolar o sobrenadante e flash congelar a -80 ° C. Lyophilize a amostra congelada por 48 horas. Armazenar as partículas liofilizadas em um dessecador a -80 ° C até o tempo de uso. </li></ol> 3. Fabricação de veículos composto de entrega (protocolo adaptado a partir do Notes VisEn Química) <ol><li> Conversão de VivoTag680 à funcionalidade superfície carboxi <ol><li> Combine um frasco de VivoTag680 com 50 mL de 1,0 M HEPES, pH 7 e anidrido succínico (2,5 mg, 25 ìmol) em 25 mL de DMSO. Adicionar 50 mL de 1,0 M NaOH a esta solução, misturado; Deixa-se reagir durante duas horas em temperatura ambiente protegido da luz. Purificar partículas usando Bio-Rad Biogel (P100, médio) com eluição com 0,1 M MES buffer, pH 6,0. Recolher o faixa verde. </li></ol></li><li> Ativação de carboxi-modificado VivoTag680 <ol><li> Combine-carboxi modificada VivoTag680 obtido em 0,1 M tampão MES, pH 6, com 1 mg de 1-etil-3-(3-dimetilaminopropil)-carbodiimida (EDC) e 2,2 mg de N-hydroxysulfosuccinimide (sulfo-NHS). Deixar reagir por 2 h à temperatura ambiente. </li><li> Purificar partículas ativado a partir agente ativador excesso (EDC) por filtração em gel usando Bio-Rad Biogel (P100, médio) com eluição com 0,1 M tampão MES, pH 6,0 e recolher a faixa verde. Conjugado imediatamente as partículas ativado para aminas contendo moléculas (por exemplo, nanopartículas BSA-carregado). Permitir que a solução para reagir por 2 horas em temperatura ambiente. Amine NPs conjugados foram purificados a partir de EDC em excesso por centrifugação a 20.000 rpm durante 60 min a 4 ° C e decantar o sobrenadante após centrifugação e ressuspensão em tampão 0,1 MES. </li></ol></li><li> Pare aqui para fabricar VivoTag680 NPs marcadas para estudos de entrega da droga. </li><li> Conjugação de carboxi-modificado PLGA-BSA-VivoTag680 nanopartículas (NP680) para microbolhas de albumina <ol><li> Combine NP680 obtido em 0,1 M tampão MES, pH 6 com 1 mg de 1-etil-3-(3 dimetilaminopropil)-carbodiimida (EDC) e 2,2 mg de N-hidroxi-sulfosuccinimide (sulfo-NHS). Permitir que a solução para reagir de uma hora à temperatura ambiente. </li><li> Purify nanopartículas ativado a partir EDC excesso por centrifugação a 20.000 rpm durante 60 min a 4 ° C e decantar o sobrenadante após a centrifugação. Ressuspender nanopartículas em 0,1 tampão MES. </li><li> Lavar MBs albumina três vezes em PBS desgaseificados para remover o excesso de solução de BSA. </li><li> Conjugado imediatamente as partículas ativado para aminas contendo moléculas (por exemplo, microbolhas de albumina). Permitir NP680 solução para reagir durante duas horas à temperatura ambiente. Purify NP680 microbolhas conjugado (MNCA) de NP680s não ligado por lavar três vezes com PBS desgaseificados. </li><li> Determinar a concentração de MNCAs usando um contador Coulter. </li></ol></li></ol> 4. Modelo de Tumor Todos os experimentos com animais estavam em conformidade com um protocolo de animais aprovados pela Universidade de Virginia Animal Care e do Comitê Use. <ol><li> C6 de ratos Giloma linhagem de células tumorais foi fornecida pelo Dr. Jason Sheehan da UVA (Charlottesville, VA). <ol><li> Linha de células foi testado para garantir células estavam livres de micoplasma. </li></ol></li><li> Manter a linha celular em F-12K Mistura de nutrientes suplementada com 16% soro de cavalo, 3% soro fetal bovino (FBS) e 1% Pen-Strep, (Gibco, EUA) a 37 ° C e 5% CO 2. </li><li> Inocular C57BLJ6/Rag1 camundongos (Jackson) com 3 x 10 6 células tumorais C6 Giloma suspensas em 300 ml de PBS por via subcutânea no membro posterior esquerdo. Permitir tumor de crescer por 12 dias para chegar a um diâmetro máximo de 8-10mm. </li></ol> 5. In Vivo Aplicação de ultra-som <ol><li> Anestesiar camundongos com uma injeção combinação intraperitoneal (IP) de cloridrato de cetamina (60 mg peso corporal / kg) e xilazina (0,1 peso corporal mg / kg) antes do tratamento. </li><li> Preparar uma manutenção anestésica de cloridrato de cetamina (20 mg peso corporal / kg) e xilazina (0,3 peso corporal mg / kg). Administrar, conforme necessário. </li><li> Canular a veia da cauda de cada animal para a administração intravenosa (IV) de um MB, MB / NP ou solução MNCA. </li><li> Tumor medições de perfusão <ol><li> IV infundir uma solução MB lipídico (1x10 8 MBs / g peso corporal em 0,3 ml de solução salina 0,9%) a uma taxa de 15μl/min com uma bomba de infusão contínua (Harvard Apparatus PHD 2000; Harvard Apparatus, Holliston, MA). </li><li> Acuson Sequoia um 512 ultra-sonografia do sistema (Siemens Medical Solutions, Mountain View, CA) equipado com um 8 13 MHz linear 15L8 sonda foi utilizada neste estudo para quantificar o contraste de perfusão do tumor. Casal sondar a 15L8 ao tumor com um gel à base de água de ultra-som (Parker Laboratories Aquasonic 100; Parker Laboratories, Inc., Fairfield, NJ). Digitalizar o tumor em B-Mode para obter o melhor plano de imagem. </li><li> Em contraste modo de pulso seqüência (CPS), adquirir um vídeo contínuo capturar microbolhas intensidade sinal de 5 segundos antes e 20 seguintes, a grande amplitude &quot;estouro&quot; de pulso em uma taxa de quadros de 13Hz. Repetir as medições em quatro planos de imagem. </li><li> Espere 10 minutos para infundir MBs albumina e iniciar o tratamento terapêutico de frequência baixa para permitir MBs de lipídios para limpar da circulação. </li></ol></li><li> Tratamento ultra-som terapêutico <ol><li> Dez minutos seguintes medidas de perfusão do tumor (5,3) um par 0,75&#39;&#39;diâmetro transdutor de 1 MHz unfocused (A314S; Panametrics, Waltham, MA) para a pele acima do tumor flanco. IV infundir um MB de albumina (1 x10 5 MBs / g peso corporal em 0,3 ml de solução salina 0,9%), MB / NP (1 x10 5 MBs / g e 0,2 NPs ug / g de peso corporal em 0,3 ml de soro fisiológico 0,9%) ou MNCA solução (1 x10 5 peso corporal MNCAs / g em 0,3 ml de solução salina 0,9%). </li><li> Tratamento de entrega de drogas <ol><li> Insonate de 60 minutos com a seqüência de pulso &quot;uma ruptura&quot; (5.4.2.2), durante uma infusão contínua de NPs, um co-injeção de MBs e NPs, ou MNCAs (5.4.1). </li><li> O &quot;1-Burst&quot; seqüência de pulsos consiste em 100 consecutivas um sinusóides MHz (ciclo de trabalho = 0,00002) cada um de 1V pico a pico de amplitude de um gerador de forma de onda (AFG-310; Tektronix, Inc., Beaverton, OR). Amplificar o sinal de forma de onda por um 55 dB RF com um amplificador de potência (ENI 3100LA; Eletrônico Navigation Industries, Richardson, TX). </li></ol></li><li> O tratamento ablativo <ol><li> Idêntica à 5.4.2.1, no entanto insonate com o &quot;5-Burst Médio&quot; ou &quot;5-Burst - Extended&quot; seqüência pulsando </li><li> A seqüência de pulsos &quot;5-Burst-Medium&quot; consiste de um sinusóides 5000 MHz (ciclo de trabalho = 0,005) cada um de 1V pico a pico de amplitude. A seqüência de pulsos &quot;5-Burst-Exteneded&quot; consiste de um sinusóides 10.000 MHz (ciclo de trabalho = 0,01) cada um de 1V pico a pico de amplitude. </li><li> Durante o curso do tempo de monitorar a temperatura do experimento tumor através da inserção de uma agulha termopar sonda (Omega T-tipo, HYP1-30-1/2-TG-60-SMPW-M) dois centímetros para o tumor. Registro medições de temperatura a cada cinco minutos. </li></ol></li></ol></li><li> Repita as medidas de perfusão do tumor, como descrito em 5,3 10 minutos após o tratamento terapêutico. </li></ol> 6. Quantificação de perfusão do tumor <ol><li> Usando o programa CPS sobre a Sequoia, entre no modo ACQ. Selecione ªregião e de interesse abrangendo o volume tumoral completa. Curvas de recuperação de perfusão será gerado da forma y = A (1 - e-βt) + C (Sadlowski 2002; Chromas 2001; Yeh 2004), que estão aptos para os dados do CPS. Avaliar β, uma medida relativa da velocidade de glóbulos vermelhos, pré-tratamento e pós-tratamento. </li><li> Exportar arquivos CPS para análise off-line. Converter os dados em arquivos avi. Ruptura de arquivos avi em seqüências de quadro a quadro da imagem. Usando Compaq Visual Fortan, ou um pacote de software similar, gerar um tempo médio de imagem de todos os quadros a partir do pulso destrutiva a 8 segundos após o pulso destrutivo. </li><li> Para determinar a área do tumor perfundidos, usando o software ImageJ, um limiar de 247 para oito o tempo bite-média CPS imagem e quantificar os pixels por cento maior no volume do tumor. Sobreposição da imagem modo-B correspondente ao momento em média imagem limiarizada definir com precisão área do tumor. </li><li> Por cento da média da área perfundidos por um período mínimo de quatro clipes de CPS para obter uma área final perfundidos em um ponto determinado momento de um tumor específico. </li><li> Levar o produto da área por cento perfundidos (6,4) e β (0,1) para determinar o fluxo de sangue em relação tumor. </li></ol> 7. Biodistribuição de nanopartículas no tumor <ol><li> Doze dias antes do tratamento com fluorescente etiquetado ratos lugar NPs em uma dieta de baixa alfasprot (Harlan, Indianapolis IN) para reduzir a autofluorescência causada por murinos normais quando imagem. </li><li> Raspar o local de imagem (flanco para o fígado) para remover todo o cabelo. </li><li> Remover pêlos residual com um agente de remoção química de cabelo, como Nair. <ol><li> Enxágüe de todos os removedor químico residual de cabelo ou uma queimadura química pode ocorrer. </li></ol></li><li> Camundongos imagem no sistema FMT (VisEn Medical) horas antes do tratamento e 0, 1, 4, e 24 após o tratamento, conforme descrito em 5.4.2. </li><li> Antes de ratos imagem anestesiar com uma injeção IP combinação de cloridrato de cetamina (60 mg peso corporal / kg) e xilazina (0,1 peso corporal mg / kg). Colocar o mouse sobre o cartucho de imagem. Adquirir imagens de reflectância em luz branca e modos fluorescente. Realizar fluorescentes imagem tomográfica no canal VT680. </li><li> Empregar a FMT software para gerar reconstruções em 3D dos dados de imagens utilizando uma equação normalizada Born. Após a reconstrução, selecione volumes de interesse (VOI) pelo desenho de uma região de interesse (ROI) em todos os 3 planos de imagem (X, Y, Z). Um valor médio fluorescentes e volume total e concentração VOI fluorocromo, devido a fluorescente etiquetado BSA NPs, será gerada para VOI, que abrangeu os pés e fígado. </li></ol> 8. Taxa de crescimento do tumor <ol><li> Avaliar o volume tumoral tomando medidas diárias usando calibres digital. Calcular volumes tumor usando uma aproximação elipsóide; V = 1 / 6 abc π. Onde a, b e c são os diâmetros máximos do tumor medido em três planos ortogonais. </li></ol> 9. Processamento e Análise de tumor <ol><li> Sete dias após os animais euthanize tratamento. Canular o ventrículo esquerdo e sangue desangrar com 10ml de 2% heparinizado Tris CaCl 2 Buffer (0,68 mM) de perfusão, seguido de 10ml Tris CaCl 2 Buffer (0,68 mM), cada um por 10 minutos a 100 mmHg. </li><li> Perfusão fix-tecido com uma infusão de paraformaldeído 4% em PBS (4 ° C) por 10 minutos a 100 mmHg. Deixar a amostra para corrigir por 60 minutos. </li><li> Espécimes especiais de consumo, incorporar em parafina, e cortados em cinco micro-secções. </li><li> Realização da coloração hematoxilina-eosina padrão para avaliar as alterações histológicas que podem ter ocorrido como resultado das exposições ultra-som. </li><li> Para detectar células apoptóticas, use um terminal transferase deoxynucleotidyl mediada deoxiuridina trifosfato de rotulagem final nick (TUNEL) ensaio (ApoptTag kit, Intergen Co., Norcross, GA, EUA). </li></ol> 10. Resultados representante 1. Fabricação de nanopartículas (2.0) <ol><li> Se este protocolo é pré-formado adequadamente NPs será esférico na forma, conforme determinado pela microscopia eletrônica de varredura (MEV), e possuem uma distribuição gaussiana em torno de 100 nm, conforme determinado por técnicas de espalhamento de luz. Resultados representativos são mostrados na Figura 1. </li></ol> 2. Entrega de drogas específicas (5.4.1) <ol><li> Com base em estudos publicados realizados em nosso laboratório (Song 2002, Chappell 2008) e resultados inéditos presentes na Figura 2, ultra-sônicos resultados destruição das microbolhas no aumento de liberação de nanopartículas intravascular para o tecido do tumor. Também mostramos que o nosso MNCA resultados entrega técnica na entrega de nanopartículas elevada imediatamente após o tratamento. </li></ol> 3. Ablação tumor (5.4.3) <ol><li> Nós mostramos que a destruição de ultra-sons prolongados de microbolhas, em ciclos de trabalho relativamente baixa (0,005-0,01), resulta na ablação mecânica domicrovasculatura tumor e regressão do crescimento tumoral. Se este protocolo for realizado adequadamente se deve esperar mudanças em (i) taxa de crescimento tumoral (Figura 3), (ii) a hemodinâmica do tumor (Figura 4), (iii) área de necrose, e (iv) a apoptose. </li></ol><img src="/files/ftp_upload/2145/2145fig1.jpg" alt="Figura 1" /> Figura 1. Caracterização de nanopartículas Plaga tendo BSA. (A) Imagem SEM das nanopartículas fabricadas corretamente. (B) Imagem SEM de nanopartículas indevidamente fabricada. <img src="/files/ftp_upload/2145/2145fig2.jpg" alt="Figura 2" /> Figura 2. (A) tomografia de fluorescência mediada (FMT) imagens de ultra-som tratados (em cima) e controle tratado (baixo) gliomas subcutânea imediatamente após o tratamento com um MNCAs, onde as nanopartículas (NPs) estão dando sinais de fluorescência 680 são sobrepostos em tons de cinza planar imagem de luz de excitação. <img src="/files/ftp_upload/2145/2145fig3.jpg" alt="Figura 3" /> Figura 3. Fold Representante mudança no crescimento do tumor após insonação microbolhas com a explosão de cinco protocolo estendido pulsante. <img src="/files/ftp_upload/2145/2145fig4.jpg" alt="Figura 4" /> Figura 4. B-Mode (A, C) e com contraste ultra-sonografia (B, D) imagens de um tumor subcutâneo glioma C6 em um mouse. Na imagem de pré-tratamento inicial (A) o limite do tumor em sua maioria hipoecóicas foi traçada em azul. Valorização média de tempo após a injeção intravenosa de um agente de contraste é mostrado em (B) pré-tratamento. Pós-tratamento, antes da injeção de contraste, o tumor está novamente em sua maioria hipoecóico (D). Após a injeção de contraste, há aumento significativamente menor no tumor.

<ol>
	<li>Chappell, J., Song, J., Burke, C., Klibanov, A., Price, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+delivery+of+nanoparticles+bearing+fibroblast+growth+factor-2+by+ultrasonic+microbubble+destruction+for+therapeutic+arteriogenesis.">Targeted delivery of nanoparticles bearing fibroblast growth factor-2 by ultrasonic microbubble destruction for therapeutic arteriogenesis.</a> Small. 10, 1769-1777 (2008).</li><li>Chomas, J. E., Pollard, R., Wisner, E., Ferrara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subharmonic+Phase-Inversion+for+Tumor+Perfusion+Estimation.">Subharmonic Phase-Inversion for Tumor Perfusion Estimation.</a> IEEE. 2, 1713-1716 (2001).</li><li>Davda, J., Labhasetwar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+nanoparticle+uptake+by+endothelial+cells.">Characterization of nanoparticle uptake by endothelial cells.</a> Int J Pharm. 233, 51-51 (2002).</li><li>Hynynen, K., McDannolod, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+MR+imaging-guided+focal+opening+of+the+blood-brain+barrier+in+rabbits.">Noninvasive MR imaging-guided focal opening of the blood-brain barrier in rabbits.</a> Radiology. 220, 640-646 (2001).</li><li>McDannold, N., Vykhodtseva, N., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubble+contrast+agent+with+focused+ultrasound+to+create+brain+lesions+at+low+power+levesl:+MR+imaging+and+histological+study+in+rabbits.">Microbubble contrast agent with focused ultrasound to create brain lesions at low power levesl: MR imaging and histological study in rabbits.</a> Radiology. 241, 95-106 .</li><li>McDannold, N., Vykhodtseva, N., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+the+blood-brain+barrier+with+focused+ultrasound:+Association+with+cavitation+activity.">Targeted disruption of the blood-brain barrier with focused ultrasound: Association with cavitation activity.</a> Phys Med Biol. 51, 793-807 .</li><li>McDannold, N., Clement, G. T., Black, P., Jolesz, F., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcranial+magnetic+resonance+imaging-+guided+focused+ultrasound+surgery+of+brain+tumors:+initial+findings+in+3+patients.">Transcranial magnetic resonance imaging- guided focused ultrasound surgery of brain tumors: initial findings in 3 patients.</a> Neurosurgery. 66, 323-332 (2009).</li><li>Meairs, S., Alonso, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+microbubblesand+the+blood+brain+barrier.">Ultrasound microbubblesand the blood brain barrier.</a> Progr Biophys Mol Biol. 93, 354-362 (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Optison, products insert. , (2009).</li><li>Sadlowskie, A., Chromas, J., Pollard, R., Bloch, S., Griffey, S., Wisner, W., Ferrara, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mean+Flow+Rate+and+Intergrated+Perfusion+Estimates+Obtained+with+Contrast-Assisted+Ultrasound.">Mean Flow Rate and Intergrated Perfusion Estimates Obtained with Contrast-Assisted Ultrasound.</a> IEEE Ultrasonics Symposium. , 1977-1980 (2002).</li><li>Song, J., Chappell, J. C., Qi, M., VanGieson, E. J., Kaul, S., Price, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+injection+site,+microvascular+pressure+and+ultrasound+variables+on+microbubble-mediated+delivery+of+microspheres+to+muscle.">Influence of injection site, microvascular pressure and ultrasound variables on microbubble-mediated delivery of microspheres to muscle.</a> J Am Coll. Cardiol. 39, 726-731 (2002).</li><li>Sheikov, N., McDannold, N., Vykhodtseva, N., Jolesz, F., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+of+blood-brain+barrier+opeing+induced+by+ultrasound+in+the+presences+of+microbubbles.">Cellular mechanisms of blood-brain barrier opeing induced by ultrasound in the presences of microbubbles.</a> Ultrasound Med. Biol. 30, 979-989 (2004).</li><li>Tanter, J., Fink, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focusing+and+steering+through+absorbing+and+aberrating+layers:+Application+to+ultrasonic+propagation+through+the+skull.">Focusing and steering through absorbing and aberrating layers: Application to ultrasonic propagation through the skull.</a> J Acoust Soc Am. 103, 2403-2410 (1998).</li><li>Yeh, C. K., Kruse, D. E., Lim, M. C., Redline, D. E., Ferrara, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+High+Frequency+Destruction/Reperfusion+System.">A New High Frequency Destruction/Reperfusion System.</a> IEEE. 1, 433-436 (2003).</li></ol>

Não há conflitos de interesse declarados.

Passos crítica Canulação da veia da cauda do rato: Injeção intravenosa na veia da cauda do rato pode ser um processo desafiador. No entanto, um cateter de veia da cauda pode melhorar significativamente a probabilidade de uma injeção de sucesso. Para fazer o cateter, repetidamente dobrar frente e para trás uma agulha de calibre 25 até que quebre a partir do hub. Insira a extremidade sem corte na extremidade do tubo PE 20 e selar a conexão com cola de silicone...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>ApoptTag kit</td>
<td>&nbsp;</td>
<td>Intergen Co.</td>
<td>S7110</td>
<td>&nbsp;</td>
<tr>
<td>un-capped 85:15 poly(lactic-co-glycolic acid) (PLAGA)</td>
<td>&nbsp;</td>
<td>Lakeshore Biomaterials</td>
<td>Custom</td>
<td>&nbsp;</td>
<tr>
<td>Vivo Tag 680</td>
<td>&nbsp;</td>
<td>VisEn Medical</td>
<td>10120</td>
<td>Used to Tag BSA</td>
</tr>
<tr>
<td>Poly(vinyl alcohol)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>363136</td>
<td>&nbsp;</td>
<tr>
<td>MicroTip Sonicator</td>
<td>&nbsp;</td>
<td>Misonix</td>
<td>S-4000</td>
<td>&nbsp;</td>
<tr>
<td>Sequoia</td>
<td>&nbsp;</td>
<td>Simons Medical</td>
<td>P.O.A</td>
<td>Equipped with CPS </td>
</tr>
<tr>
<td>FreeZone 2.5</td>
<td>&nbsp;</td>
<td>Labconco</td>
<td>7670020</td>
<td>Equipped with Nitrogen Trap</td>
</tr>
<tr>
<td>Methylene chloride (CH2Cl2)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>D37-500</td>
<td>&nbsp;</td>
<tr>
<td>FMT 250</td>
<td>&nbsp;</td>
<td>VisEn Medical</td>
<td>P.O.A</td>
<td>&nbsp;</td>
<tr>
<td>F-12K Nutrient Mixture</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>21127-022</td>
<td>&nbsp;</td>
<tr>
<td>polyethyleneglycol-40 stearate </td>
<td>&nbsp;</td>
<td>Sigma Chemical </td>
<td>9004-99-3</td>
<td>&nbsp;</td>
<tr>
<td>distearoyl phosphatidylcholine</td>
<td>&nbsp;</td>
<td>Avanti Polar Lipids</td>
<td>770365</td>
<td>&nbsp;</td>
<tr>
<td>Multisizer Coulter Counter</td>
<td>&nbsp;</td>
<td>Beckman Coulter </td>
<td>P.O.A</td>
<td>&nbsp;</td>
<tr>
<td>Waveform Generator </td>
<td>&nbsp;</td>
<td>Tektronix, Inc.</td>
<td>AFG-310</td>
<td>&nbsp;</td>
<tr>
<td>water-based ultrasound gel </td>
<td>&nbsp;</td>
<td>Parker Laboratories</td>
<td>Aquasonic 100</td>
<td>&nbsp;</td>
<tr>
<td>Infusion pump </td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>Harvard Apparatus PHD 2000</td>
<td>&nbsp;</td>
<tr>
<td>1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) </td>
<td>&nbsp;</td>
<td>Pierce Biotechnology</td>
<td>25952-53-8</td>
<td>&nbsp;</td>
<tr>
<td>N-hydroxysulfosuccinimide (Sulfo-NHS)</td>
<td>&nbsp;</td>
<td>Pierce Biotechnology</td>
<td>106627-54-7</td>
<td>&nbsp;</td>
<tr>
<td>Succinic anhydride</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>603902</td>
<td>&nbsp;</td>
<tr>
<td>Power Amplifier </td>
<td>&nbsp;</td>
<td>Electronic Navigation Industries</td>
<td>ENI 3100LA</td>
<td>&nbsp;</td>
<tr>
<td>Needle Thermocouple Probe </td>
<td>&nbsp;</td>
<td>Omega</td>
<td>HYP1-30-1/2-T-G-60-SMPW-M</td>
<td>&nbsp;</td>
<tr>
<td>BioGel (P100, medium)</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>150-4170</td>
<td>&nbsp;</td>
<tr>
<td>.75&#x2019;&#x2019; diameter 1 MHz unfocused transducer</td>
<td>&nbsp;</td>
<td>Panametrics</td>
<td>A314S </td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

contrast ultrasound targeted treatment of gliomas in mice via drug-bearing nanoparticle delivery and microvascular ablation

Estamos desenvolvendo minimamente invasiva de contraste de microbolhas abordagens terapêuticas em que a permeabilização e / ou ablação da microvasculatura são controladas por diferentes parâmetros de ultra-som pulsante. Especificamente, estamos testando se tais abordagens podem ser usadas para tratar tumores cerebrais malignos através da entrega de drogas e ablação microvascular. Estudos preliminares foram realizados para determinar se a entrega de nanopartículas alvo de drogas rolamento pode ser facilitada pela destruição mediada ultra-som de &quot;composite&quot; agentes de entrega composto de 100 nm poli (ácido lático-co-ácido glicólico) (Plaga) nanopartículas que estão aderidas à albumina microbolhas sem casca . Denotamos esses agentes como agentes de microbolhas nanopartículas composto (MNCAs). Quando direcionados para subcutânea gliomas C6 com ultra-som, observamos um aumento de 4,6 vezes imediato na entrega de nanopartículas em tumores MNCA tratados sobre os tumores tratados com microbolhas co-administrado com nanopartículas e um aumento de 8,5 vezes em relação a tumores não tratados. Além disso, em aplicações de câncer de muitos, acreditamos, pode ser desejável para realizar entrega de drogas específicas em conjunto com a ablação da microcirculação tumor, o que levará à hipóxia tumoral e apoptose. Para este fim, nós testamos a eficácia da não-theramal cavitação induzida por ablação microvascular, mostrando que essa abordagem provoca redução de perfusão do tumor, a apoptose, inibição do crescimento significativo, e necrose. Em conjunto, estes resultados indicam que a nossa abordagem orientada por ultra-som tem o potencial de aumentar a eficiência terapêutica através da criação de necrose tumoral por meio de ablação microvascular e / ou simultaneamente aumentar a carga de drogas em gliomas.

Watch this Scientific Journal Video about Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation at JoVE.com

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

Insonação de microbolhas é uma estratégia promissora para a ablação do tumor em reduzido tempo médio poderes acústica, bem como para a entrega prevista para terapêutica. O objetivo do presente estudo é desenvolver estratégias de ciclo de baixa dever de ultra-som pulsante e nanocarriers para maximizar a não-térmicos ablação microvascular e entrega de carga útil para subcutânea 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 ...

contrast-ultrasound-targeted-treatment-gliomas-mice-via-drug-bearing

Research

JoVE Journal

Bioengineering

15.8K visualizações.  University of Virginia. Insonação de microbolhas é uma estratégia promissora para a ablação do tumor em reduzido tempo médio poderes acústica, bem como para a entrega prevista para terapêutica. O objetivo do presente estudo é desenvolver estratégias de ciclo de baixa dever de ultra-som pulsante e nanocarriers para maximizar a não-térmicos ablação microvascular e entrega de carga útil para subcutânea C6 gliomas.

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

Introduction to the Ultrasound Targeted Microbubble Destruction 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 microbubble contrast agents7-9. In mice these vector-carrying microbubbles can be administered intravenously or directly to the left ventricle of the heart. In larger animals they can also be infused through an intracoronary catheter. The subsequent delivery from the circulation to a target organ occurs by acoustic cavitation at a resonant frequency of the microbubbles. It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in or between the endothelial cells of the microvasculature of the targeted region10. As a result of this sonoporation effect, the transfection efficiency into and across the endothelial cells is enhanced, and transgene-encoding vectors are deposited into the surrounding tissue. Plasmid DNA remaining in the circulation is rapidly degraded by nucleases in the blood, which further reduces the likelihood of delivery to non-sonicated tissues and leads to highly specific target-organ transfection.

Ultrasound Targeted Microbubble Destruction (UTMD) can be used to direct site-specific delivery of bioactive molecules, including therapeutic genes, to target organs accessible to ultrasound, such as the heart and liver1-6.

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 ...

Bioengenharia

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

Extração de lipídeos celulares para Targeted diluição de isótopos estáveis ​​Líquido Análise Espectrometria de Massa-Cromatografia

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

Entrega de genes e Transfection em células pancreáticas humanas Cancer usando Fator de Crescimento Epidérmico Receptor-alvo à base de gelatina Nanovectors Engineered

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

A microfluídica Endothelialized para estudo das interacções microvasculares em Doenças Hematológicas

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Avaliação de Entrega Gene Polimérico Nanopartículas por Análise Nanoparticle Rastreamento e alta capacidade de Citometria de Fluxo

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

Monitoramento em tempo real de High Intensity Focused Ultrasound (HIFU) Ablação de<em&gt; In Vitro</em&gt; Canine Fígados Usando Movimento Harmônico Imaging para ultra-som focalizado (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

Targeted Plasma Membrane entrega de um hidrofóbica Carga encapsulado em um cristal líquido nanopartículas Transportador

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

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 ...

Patient-Specific Polyvinyl Alcohol Phantom Fabrication with Ultrasound and X-Ray Contrast for Brain Tumor Surgery Planning

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create anatomically accurate head phantoms with realistic brain imaging properties because standard fabrication methods are not optimized to replicate any patient-specific anatomical detail and 3D printing materials are not optimized for imaging properties. In order to test and validate a novel navigation system for use during brain tumor surgery, an anatomically accurate phantom with realistic imaging and mechanical properties was required. Therefore, a phantom was developed using real patient data as input and 3D printing of molds to fabricate a patient-specific head phantom comprising the skull, brain and tumor with both ultrasound and X-ray contrast. The phantom also had mechanical properties that allowed the phantom tissue to be manipulated in a similar manner to how human brain tissue is handled during surgery. The phantom was successfully tested during a surgical simulation in a virtual operating room.
The phantom fabrication method uses commercially available materials and is easy to reproduce. The 3D printing files can be readily shared, and the technique can be adapted to encompass many different types of tumor.

This protocol describes the fabrication of a patient specific skull, brain and tumor phantom. It uses 3D printing to create molds, and polyvinyl alcohol (PVA-c) is used as the tissue mimicking material.

Fabricação fantasma do álcool polivinyl específico do paciente com ultrassom e contraste de raios-X para planejamento de cirurgia de tumor cerebral

Phantoms are essential tools for clinical training, surgical planning and the development of novel medical devices. However, it is challenging to create ...