Este trabalho foi financiado por subvenções a LKM-K dos Institutos Nacionais de Saúde / Instituto Nacional do Câncer (R01CA129822 e R01CA140995). Dr. Medina-Kauwe graças C. Rey, M. M-Kauwe e D. Revetto para apoio contínuo.

1. Entrega sistêmica In vivo <ol><li> Misturar suficiente Herdox com solução salina estéril para equiparar 0,2 ml de uma dose de 0,004 mg / kg de Herdox por injecção durante 6-8 semanas de idade nu / nu rato rolamento tumores subcutâneos bilaterais xenoenxerto flanco. </li><li> Suavemente Herdox desenhar a mistura para uma seringa de insulina 3/10 cc com uma agulha 29G, evitando bolhas. </li><li> A anestesia é induzida por exposição breve isoflurano numa câmara de indução equipado com um sistema de limpeza de gás (taxa de fluxo de oxigénio: 0,5-1 l / min, concentração de isoflurano: 3-4% (ou inferior). </li><li> Injectar a mistura total na veia da cauda de um rato anestesiado (0,2 ml por injecção). Injecção IV também pode ser realizada em um contido, rato não anestesiados. </li><li> Repetir a injecção no mesmo rato para mais seis dias seqüenciais, uma vez por dia. </li></ol> 2. Imagens de fluorescência in vivo O acúmulo de Herdox fluorescência em tumores podeser detectáveis ​​até ao último dia da injecção (Dia 7), utilizando um gerador de imagens multimodo. Os procedimentos abaixo implica a utilização de uma macro-sistema de iluminação e de detecção de personalização (Figura 1) 8. <ol><li> Ligue o multimodo In vivo Imager Optical. </li><li> Escolha um filtro passa-banda de emissão (590 nm ± 30 nm) adequado para a detecção de fluorescência doxorrubicina. </li><li> Ligue Argon-Krypton laser e colocar um filtro passa-banda de excitação (488 nm ± 10 nm) no caminho óptico laser. </li><li> Ligue o sistema de anestesia e em seguida, coloque o mouse na câmara de anestesiante (taxas de fluxo de oxigênio: 0,5-1 L / min, concentração de isoflurano: 3-4% (ou inferior), equipados com um sistema de limpeza de gás. </li><li> Transferir o mouse da câmara de anestesiar à câmara de imagem do multimodo In vivo Imager Optical quando o mouse está anestesiado. </li><li> Coloque um nosecone sobre o nariz do mouse e abrir o fluxo de administrar anestesia contínuasia durante a aquisição de imagem (taxa de fluxo de oxigênio: 0,5-1 L / min, a concentração de isoflurano: 2-3% (ou inferior). </li><li> Adquirir imagens de fluorescência utilizando um tempo de exposição de 5-15 seg. </li><li> Realizar a análise de imagem e processamento, incluindo correção de fundo ou ajuste de contraste. </li></ol> 3. Imagem de Fluorescência ex vivo Herdox fluorescência pode ser trabalhada em tumores e órgãos específicos (incluindo o fígado, rim, baço, coração e músculo esquelético), colhidas a partir de ratinhos sacrificados às 24 horas após a última injecção (7 dias) de Herdox. <ol><li> Ligue o multimodo In vivo Imager Optical. </li><li> Escolha um filtro passa-banda de emissão (580 nm ± 20 nm) para detecção de fluorescência doxorrubicina. </li><li> Ligue Argon-Krypton laser e colocar um filtro passa-banda de excitação (488 nm ± 10 nm) no caminho óptico laser. </li><li> Tumores lugar e órgãos específicos dispostos em uma placa de Petri para a imagem da câmara of o multimodo Em Imager Optical vivo. </li><li> Adquirir imagens de fluorescência de tecidos, utilizando um tempo de exposição de 5-15 seg. Um exemplo de aquisição da imagem de fluorescência inicial é mostrado na figura 2a. Repita o mesmo usando uma placa de Petri vazia, que servirá como pano de fundo (Figura 2b). </li><li> Realizar a análise de imagem e processamento, incluindo correção de fundo ou ajuste de contraste. Um exemplo de uma imagem corrigida é mostrado na Figura 2c, resultante da subtração da Figura 2b a partir da Figura 2a. </li></ol> 4. In situ imagem confocal de Tumores Na imagem confocal situ permite a detecção e análise da acumulação tumoral Herdox ao nível celular. <ol><li> Ligue um microscópio confocal Leica SPE. </li><li> Selecione 488 luz laser nm para a excitação de doxorrubicina e emissão de comprimentos de onda (560-620 nm) para a doxorrubicinadetecção por fluorescência. </li><li> Selecione uma objetiva de 40X ou 63X e óleo de imersão queda na lente objetiva. </li><li> Extrair tumores fresco a partir de ratos sacrificados que receberam previamente tratamentos Herdox e simulada como descrito no procedimento 2. </li><li> Tumores colocar em uma placa de Petri sobre gelo para evitar a degradação do tecido e, em seguida, transferir os tumores a uma câmara Delta T para a imagem confocal. </li><li> Adquirir imagens confocal dos tumores em profundidades focais seqüenciais (tamanho do passo: 1 mícron, espessura: 20 mm). Um exemplo de imagens adquiridas sequencialmente ao longo do eixo z é mostrado na Figura 3, painel da esquerda. </li><li> Realizar intensidade máxima z projecção das imagens. Uma projecção de intensidade máxima z empilhado imagens é mostrado na Figura 3, painel da direita. </li><li> Calcular média intensidades de fluorescência de intensidade máxima Z -. Imagens de projeção média intensidades de fluorescência das imagens sobre o campo de vista global foram calculard usando ImageJ. </li></ol> 5. Spectral Imaging Ratiometric e Análise Imagem espectral Ratiometric e análise permite discriminação entre Dox fluorescência e autofluorescência. <ol><li> Energia em um microscópio confocal de varredura a laser de fluorescência. </li><li> Adquirir 15 imagens dos tumores Herdox-tratadas e não tratadas, a uma profundidade especificada no intervalo espectral de 510-650 nm, com um tamanho de passo de 10 nm, e a luz de excitação a 488 nm, utilizando um microscópio confocal Leica SPE. </li><li> Prepara-se uma solução 100 uM de doxorrubicina. </li><li> Realizar imagem espectral da solução de doxorrubicina 100 uM para obter a assinatura espectral pura de fluorescência Dox (faixa espectral: 510-650 nm, um tamanho de passo: 10 nm). Os resultados típicos de aquisição de imagem a partir de imagiologia espectral e do espectro de fluorescência resultante representada em gráfico são mostrados na Figura 4. </li><li> Adquirir a assinatura espectral autofluorescência de uma imagem cube (faixa espectral: 510-650 nm, um tamanho do passo: 10 nm) obtidos por imagem espectral de tumores não tratados. Os resultados típicos de aquisição de imagem a partir de imagiologia espectral e do espectro de fluorescência resultante representada em gráfico são mostrados na Figura 5. </li><li> Gerar quatro assinaturas espectrais de referência (puro autofluorescência, 0.1.doxorubin 0,9. Autofluorescência, 0.2. Doxorrubicina 0,8. Autofluorescência, 0.3.doxorubin 0,7. Autofluorescência) utilizando o programa que nós desenvolvemos 9. Uma curva típica que mostra quatro assinaturas espectrais de referência é mostrado na Figura 6. </li><li> Realizar classificação espectral das imagens, tal como definido pela referência de assinaturas espectrais através de medida de distância euclidiana usando o programa que anteriormente desenvolvido 9. </li><li> Realizar separação espectral linear dessas imagens usando um programa espectral desmistura (plug-in ImageJ) desenvolvemos 7, 10, para comparação com a imagem espectral e análise raciométrica. Um example de separar fluorescência Herdox de autofluorescência por separação espectral linear é mostrada na Figura 7. </li></ol>

<ol>
	<li>Agadjanian, H., Chu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+Targeting+by+DNA+Capture+in+Viral+Protein+Particles.">Chemotherapy Targeting by DNA Capture in Viral Protein Particles.</a> Nanomedicine. 7 (3), 335-352 (2012).</li><li>Agadjanian, H., Ma, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+detection+and+elimination+by+a+targeted+gallium+corrole.">Tumor detection and elimination by a targeted gallium corrole.</a> Proc. Natl. Acad. Sci. U.S.A. 106 (15), 6105-6110 (2009).</li><li>Agadjanian, H., Weaver, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+delivery+of+corroles+to+cells+via+noncovalent+conjugates+with+viral+proteins.">Specific delivery of corroles to cells via noncovalent conjugates with viral proteins.</a> Pharm. Res. 23 (2), 367-377 (2006).</li><li>Medina-Kauwe, L. K., Maguire, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-viral+gene+delivery+to+human+breast+cancer+cells+by+targeted+Ad5+penton+proteins.">Non-viral gene delivery to human breast cancer cells by targeted Ad5 penton proteins.</a> Gene Therapy. , 81753-81761 (2001).</li><li>Medina-Kauwe, L. K., Kasahara, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3PO,+a+novel+non-viral+gene+delivery+system+using+engineered+Ad5+penton+proteins.">3PO, a novel non-viral gene delivery system using engineered Ad5 penton proteins.</a> Gene Therapy. , 8795-8803 (2001).</li><li>Rentsendorj, A., Xie, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Typical+and+atypical+trafficking+pathways+of+Ad5+penton+base+recombinant+protein:+implications+for+gene+transfer.">Typical and atypical trafficking pathways of Ad5 penton base recombinant protein: implications for gene transfer.</a> Gene Ther. 13 (10), 821-836 (2006).</li><li>Hwang, J. Y., Park, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodality+Imaging+In+vivo+for+Preclinical+Assessment+of+Tumor-Targeted+Doxorubicin+Nanoparticles.">Multimodality Imaging In vivo for Preclinical Assessment of Tumor-Targeted Doxorubicin Nanoparticles.</a> PLoS ONE. 7 (4), e34463 (2012).</li><li>Hwang, J. Y., Wachsmann-Hogiu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Multimode+Optical+Imaging+System+for+Preclinical+Applications+In+Vivo:+Technology+Development,+Multiscale+Imaging,+and+Chemotherapy+Assessment.">A Multimode Optical Imaging System for Preclinical Applications In Vivo: Technology Development, Multiscale Imaging, and Chemotherapy Assessment.</a> Mol. Imaging Biol. , (2011).</li><li>Hwang, J. Y., Gross, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ratiometric+spectral+imaging+for+fast+tumor+detection+and+chemotherapy+monitoring+in+vivo.">Ratiometric spectral imaging for fast tumor detection and chemotherapy monitoring in vivo.</a> J. Biomed. Opt. 16 (6), 066007 (2011).</li><li>Fujimoto, J. G., Farkas, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomedical Optical Imaging. , (2009).</li><li>Hwang, J. Y., Moffatt-Blue, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimode+optical+imaging+of+small+animals:+development+and+applications.">Multimode optical imaging of small animals: development and applications.</a> Proc. of SPIE. 6411, (2007).</li><li>Ducros, M., Moreaux, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+unmixing:+analysis+of+performance+in+the+olfactory+bulb+in+vivo.">Spectral unmixing: analysis of performance in the olfactory bulb in vivo.</a> PLoS One. 4 (2), e4418 (2009).</li><li>Zimmermann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+imaging+and+linear+unmixing+in+light+microscopy.">Spectral imaging and linear unmixing in light microscopy.</a> Adv. Biochem. Eng. Biotechnol. , 95245-95265 (2005).</li></ol>

O autor, Daniel Farkas, é presidente do espectral Molecular Imaging. Os demais autores não têm interesses conflitantes.

Dox fluorescência pode ser detectada in vivo utilizando o gerador de imagens multimodo quando os tumores são subcutâneas. No entanto, a dose terapeuticamente eficaz de Herdox (0,004 mg / kg) é inferior ao limiar de detecção, após uma única dose. Em contraste, após 7 injecções diárias (1x/dia durante 7 dias), a acumulação do tumor e retenção das partículas é suficiente para permitir a visualização de Dox fluorescência. É essencial quando se trabalha com Dox ou q...

Tumor-alvo de quimioterapia tem o potencial de eliminar células cancerosas com uma dose mais baixa em comparação com as drogas irrelevantes porque a maior parte da terapia administrada pode acumular-se ao seu destino pretendido, em vez de distribuir a tecido não do tumor. A última situação iria diluir a eficácia do fármaco e, assim, requerem doses mais elevadas para ser eficaz, o tumor-alvo tem vantagens terapêuticas e segurança ao longo do tratamento não específico padrão. Targeting quimioterapia por encapsulamento em nanopartículas automontadas permite que a droga permaneça quimicamente não modificado, em contraste com as drogas que são covalentemente ligados a moléculas alvo. Como tal ligação tem o potencial de alterar a actividade de ambos o fármaco e a molécula alvo; montagem não covalente permite que a potência da droga a ser retida. Nós mostramos anteriormente que o romance de três componentes, complexo de auto-montados, Herdox, visa tumores HER2 + <em&gt; In vivo e provoca a ablação de tumores de crescimento, poupando o tecido normal, incluindo o coração 1. Herdox é formado através de interacções não covalentes entre a proteína de ligação ao receptor de células-penetração, HerPBK10, e o agente quimioterapêutico, a doxorrubicina (Dox), através de um pequeno ligante de ácido nucleico. HerPBK10 se liga o factor de crescimento epidérmico humano (HER), e desencadeia a endocitose mediada pelo receptor de 2-4, enquanto que a penetração da membrana endossomal é conseguida através da incorporação do derivado de adenovírus penton base de proteína da cápside 4-6. Um domínio carregado positivamente com a proteína de ligação de ácido nucleico permite a 4, 5, através da qual o ADN-Dox intercalado pode ser transportado para a entrega alvo. Eletrofílica, intercalação e, possivelmente, as interações de oligomerização da proteína facilitar a auto-montagem em volta de 10-20 nm partículas que são estáveis ​​no sangue e sob o armazenamento prolongado em temperaturas diferentes 1. Alvo preferencial para HER2 + células tumorais é facilitada pela afinidade de ligando aumentada quando HER2 é elevado. Os nossos estudos anteriores mostraram que a administração sistémica de rendimentos Herdox acumulação preferencial em tumores de mais de tecido não-tumoral e em comparação com uma Dox irrelevantes, e a penetração de células tumorais in vivo, 7. Temos observado que as versões Herdox Dox após a entrada de células tumorais, permitindo a acumulação Dox no núcleo 1. Tumor-acumulação parece correlacionar-se com o nível do receptor, como tumores que expressam HER2 relativamente baixas acumulam menos Herdox em comparação com aqueles com níveis mais elevados comparativamente HER2 1. Além disso, a concentração efectiva de morte celular exibe uma correlação inversa com a exibição de HER2 em linhas de células de tumor que expressam superfície celular diferentes níveis de HER2 1. Herdox apresenta uma vantagem terapêutica e segurança sobre Dox irrelevantes, como a matança tumor ocorre em mais de dose de 10 vezes menor em comparação com a untarigualmente dirigido droga e não produz efeitos adversos detectáveis ​​no coração (detectado por ecocardiografia e histológica mancha) ou fígado (detectado por coloração TUNEL) do tecido, em contraste com um Dox irrelevantes. Apesar de sua derivação a partir de uma proteína do capsídeo viral, HerPBK10 não apresenta imunogenicidade detectável em níveis terapêuticos 2. Considerando anticorpos pré-existentes para adenovírus todo pode reconhecer HerPBK10, eles são incapazes de impedir a ligação de 2 celular. O volume do tumor medido ao longo do tempo é um método padrão de avaliação da eficácia terapêutica de terapêuticas específicas, e tem sido utilizada para avaliar a eficácia terapêutica do Herdox. Complementando esta abordagem com in vivo e ex vivo de imagens intensidade de fluorescência nos permitiu avaliar melhor direcionamento eficiência 7. Temos especificamente integrados in situ de imagem confocal de tumores excisados ​​com a análise espectral de Dox fluorescência para verificar que nenhum Herdoxt só acumulou em tumores in vivo, mas penetrou nas células tumorais e Dox entregue no citoplasma e núcleo 7. A análise espectral, além disso, nos permitiu distinguir Dox fluorescência da autofluorescência 7. Aqui demonstramos em maior detalhe a nossa abordagem para avaliar Herdox in vivo, após a administração sistémica, e mais importante, para avaliar a segmentação por métodos de imagem multimodo e análises.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</th
 ></tr>
 <tr>
 <td >Fluorescence laser scanning confocal microscope</td>
 <td >Leica</td>
 <td >SPE</td>
 <td></td>
 </tr>
 <tr>
 <td>In Vivo Optical Imager</td>
 <td>Spectral Molecular Imaging</td>
 <td>Multimode In Vivo Optical Imager</td>
 <td></td>
 </tr>
 <tr>
 <td >Doxorubicin-HCl</td>
 <td >Sigma-Aldrich</td>
 <td >D4035</td>
 <td></td>
 </tr>
 <tr>
 <td >Nude (NU/NU) mouse, female, 6-8 week</td>
 <td >Charles River</td>
 <td >Strain code 088</td>
 <td></td>
 </tr>
 <tr>
 <td >MDA-MB-435 human HER2+ tumor cells</td>
 <td >NCI-Frederick Cancer DCTD Tumor/Cell Line Repository</td>
 <td >0507292</td>
 <td></td>
 </tr>
 <tr>
 <td >3/10 cc insulin syringe U-100 with 29G x 1/2" Ultra-FineIV permanently attached needle</td>
 <td >BD</td>
 <td >309301</td>
 <td></td>
 </tr>
 <tr>
 <td >Delta T chamber </td>
 <td >Bioptechs </td>
 <td >04200417B</td>
 <td></td>
 </tr>
 </tbody>
</table>

analysis of targeted viral protein nanoparticles delivered to her2+ tumors

A nanopartícula HER2 + tumor-alvo, Herdox, apresenta acúmulo tumor preferencial e ablação de tumores de crescimento em um modelo animal de câncer HER2 +. Herdox é formada por auto-montagem não covalente de uma proteína de penetração nas células alvo do tumor com o agente de quimioterapia, doxorubicina, através de um pequeno ligante de ácido nucleico. Uma combinação de eletrofílico, intercalação e interações oligomerização facilitar a auto-montagem em partículas redondas 10-20 nm. Herdox exibe estabilidade no sangue, bem como em armazenamento prolongado a temperaturas diferentes. Entrega sistémica de Herdox em ratos portadores de tumor resulta na morte de células de tumor, sem efeitos adversos detectáveis ​​para tecido não-tumoral, incluindo o coração e fígado (que sofrer danos marcados por doxorubicina não segmentados). HER2 elevação facilita direccionamento para células que expressam o receptor do factor de crescimento epidérmico humano, daí tumores exibindo níveis elevados de HER2 exibem uma maior acumulação de células em comparação com Herdox expressãocantar níveis mais baixos, tanto in vitro como in vivo. Imagem intensidade de fluorescência combinada com a análise espectral confocal e situ nos permitiu verificar em tumor vivo segmentação e penetração de Herdox após a entrega sistemática de células tumorais. Aqui detalhamos os nossos métodos para avaliar tumor alvo através de imagens multimodo após a entrega sistêmica.

A Figura 1 mostra o gerador de imagens in vivo protótipo óptico, o qual foi construído com o objectivo de aquisição de imagem em várias modalidades, incluindo a intensidade de fluorescência, espectral, tempo de vida, 2-fotão, confocal intra-vital, e imagem de bioluminescência. Além disso, a elevada sensibilidade da câmara de refrigeração e as linhas de laser de alta potência incorporado no presente sistema produz imagens de fluorescência de contraste mais elevados em comparação...

Watch this Scientific Journal Video about Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors at JoVE.com

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

Este artigo detalha os procedimentos para a análise das nanopartículas tumor-alvo, Herdox imageamento óptico. Em particular, o uso detalhado do aparelho de imagem para detecção do tumor multimodo alvo e avaliar a penetração do tumor é descrito aqui.

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. ...

analysis-targeted-viral-protein-nanoparticles-delivered-to-her2

Research

JoVE Journal

Bioengineering

10.9K visualizações.  University of Southern California. Este artigo detalha os procedimentos para a análise das nanopartículas tumor-alvo, Herdox imageamento óptico. Em particular, o uso detalhado do aparelho de imagem para detecção do tumor multimodo alvo e avaliar a penetração do tumor é descrito aqui.

Vídeo: Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

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

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 ...

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. ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

Nanopartículas virais para<em&gt; Em vivo</em&gt; Imaging Tumor

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

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 ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

Harmonic Nanopartículas Regenerativa Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

Produção de nanopartículas metálicas por ablação a laser pulsada em líquidos: uma ferramenta para estudar as propriedades antibacterianas das nanopartículas

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Correlativo luz microscopia eletrônica de varredura (CLEM) para rastreamento e Imaging proteína Viral associado estruturas nas células Cryo-imobilizado

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKC&#948;i. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

Proteína quinase C-delta inibidor do Peptide formulação utilizando nanopartículas de ouro

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a ...