Este trabalho foi financiado pelo NIH (U54CA143837), o NIH MD Anderson Cancer Center, Suporte Grants (CA016672), a Fundação V (SAC), e uma bolsa de investigação irrestrito da Fundação Kanzius Pesquisa (SAC, Erie, PA). Agradecemos Kristine Ash, do Departamento de Oncologia Cirúrgica, MD Anderson Cancer Center, de assistência administrativa.

Uma visão geral experimental completo está representado na Figura 1. Mais detalhes são descritos nas etapas 1-3 abaixo. 1. Avaliando Aquecimento RF de NP Colóides: AuNPs como exemplo <ol><li> Em geral, para cada amostra a ser investigada NP, primeiro lavar várias vezes as amostras por meio de um filtro de centrifugação com água desionizada (DI) de água para remover os iões de fundo e contaminantes. Todos os iões e contaminantes terá sido removida da suspensão AUNP quando o líquido a ser lavado para fora tem taxas semelhantes de aquecimento RF (HR) como água DI. Este processo de purificação permite também que para as concentrações mais elevadas de PN a ser obtidos. Vale a pena notar que, embora utilizando AuNPs neste exemplo, os princípios fundamentais podem ser aplicados a outros materiais NP. </li><li> Como um exemplo, purificar um frasco de 500 ml de AuNPs disponíveis comercialmente de diâmetro de 5 nm e, em seguida, submetê-los a um campo de RF de 13,56 MHz de eletro-field força de 90 kV / m. <ol><li> Tome ~ 125 ml da solução estoque AUNP e divisão entre seis a 50 kDa tubos de filtro de centrífuga. Centrifugar a 3.000 rpm. durante 2 min 5 seg. Remover filtros de buffer e de recarga filtrados com mais solução estoque. Repita até que todos os 500 ml foi filtrada. </li><li> Substituir o tampão filtrado com um volume similar de água DI e repetir cerca de 8 vezes (ou até que as HRs RF tampão filtrados são equivalentes a água Dl). Nota, a análise por UV-Vis, também pode ser utilizado para monitorar os picos de absorção de contaminantes. Uma vez que os contaminantes tampão ter sido totalmente removido, pipeta de aproximadamente 0,5 ml de água Dl para cada filtro e ressuspender as AuNPs por pipetagem repetida. Isto deve remover completamente os AuNPs do filtro e para permitir a ressuspensão completa. Misture todos os seis suspensões em um tubo de 15 ml Eppendorf. </li><li> Uma vez que os AuNPs foram purificadas e concentradas, analisar a amostra utilizando ICP-OES e / ou ICP-MS, UV-vis e potencial Zeta de dados sobre concentration e estabilidade NP, respectivamente. SEM e / ou análise de TEM, também pode ser utilizado para obter os dados morfológicos. A preparação da amostra detalhada para estas técnicas podem ser encontrados na literatura 4. </li></ol></li><li> Usando o sistema de Kanzius RF descrito em estudos anteriores 8, ou derivações deste sistema, coloque um 1,3 ml cilíndrico quartzo tina de modo que o campo elétrico RF no ar (sem amostra presente) seria a 90 kV / m no interior da cuba. Para uma amostra de soro fisiológico normal (0,9% NaCl) a-campo eléctrico será reduzida para ~ 1,1 kV / m. Estas são as condições aproximadas utilizadas para permitir que sejam feitas comparações entre diferentes sistemas. <ol><li> Pipetar 1,3 ml de uma amostra de 1000 mg / l de colóide AUNP purificada na cuvete de quartzo e introduzi-la em a-campo RF. Isto pode ser feito por meio de um suporte de amostra de Teflon construído sob encomenda. Expor amostra para o campo de RF durante um período de 120 segundos ou até a amostra atingir 70 ° C para evitar a formação de arco eléctrico ou de ebulição rápida. Capture os dados de imagens térmicas (bem como as áreas de controle) usando uma câmera IR e software associado. Repita este procedimento três vezes. </li><li> Filtrar a amostra através de um outro filtro de centrífuga de 50 kDa para extrair os AuNPs do tampão de água DI. Re-expor o tampão para o campo de RF, novamente três vezes. A diferença de horas entre o colóide AUNP eo buffer de água DI fundo determina o HR devido aos próprios AuNPs. Esperar obter horas de ~ 0,3 ° C / seg e 0,05 ° C / seg a dar um AUNP dependente de HR de ~ 0,25 ° C / seg. Volte a suspender as AuNPs restantes do filtro em 1,3 ml de água para experimentos in vitro in vivo /. </li></ol></li></ol> 2. Hipertermia induzida pela RF assistida por Nanopartículas: Estudos in vitro <ol><li> Estes estudos in vitro, pode ser aplicado a qualquer tipo de tipo de célula cancerosa que formam monocamadas 2D. Neste experimento usar carcinoma hepatocelular humano derivado de células Hep3B. <ol><li> Placa ~ 50.000 cells nas três dianteiros cavidades de uma placa de 12 poços com 1 ml de meio de crescimento. Repita este 6 vezes (usar três placas para estudos NP e três placas como controles). Incubar a 37,5 ° C por 24 horas antes de introduzir os NPs. Esterilizar PN primeiro usando BioSafety Gabinete exposição UV-luz por 5 min. </li><li> Em cada poço introduzir 0,1 ml de uma solução 1,000 mg / L AUNP e deixar durante mais 24 horas. Adicionar 0,1 ml de água em cada poço das placas de três células de controlo e também deixar durante 24 horas. </li><li> Após 24 horas ter passado, aspirar os meios de comunicação das células e lavagem com PBS para remover quaisquer AuNPs ligadas à superfície. Substitua a mídia celular. As células estão prontas para a exposição RF. </li></ol></li><li> Coloque cada pacote de células de 12 poços dentro do campo de RF. Espere até que as células tenham arrefecido a 31 ° C. Ligue o gerador de RF e expor para 3,5 min. A temperatura final do meio celular será ~ 37 ° C. Desligue o campo de RF. Remover as células e colocá-los em uma incubadora por 24 horas antes da análise. <ol> &lt;li&gt; Retirar células da incubadora e media célula aspirado. Adicionar 1,6 ml de meio de células de cada poço, assim, de 0,4 ml de reagente MTT. Incubar as células durante 4 horas. Aspirar a mídia e substituir com 2 ml de dimetilsulfóxido (DMSO). Colocar as placas da célula num agitador de bancada e deixar durante 10 minutos para permitir que o DMSO, para solubilizar os reagentes MTT. Finalmente, uma pipeta de 100 uL de cada poço para uma placa de 96 poços e opticamente lido do poço a 570 nm utilizando um leitor de placas, tais como o leitor de placas SPECTROstar Nano. </li></ol></li></ol> 3. Hipertermia induzida pela RF assistida por Nanopartículas: estudos in vivo <ol><li> Estes estudos in vivo, pode ser aplicado a qualquer tipo de cancro que formam tumores sólidos, em um modelo murino ortotópico ou ectópica. Este experimento utiliza células de câncer de fígado Hep3B em um modelo de camundongo ectópica tumor BALB-C Nude. </li><li> Nota: todos os experimentos in vivo são executadas em conformidade com todas as diretrizes, regulamentos e agências reguladoras. Além disso, o protocolo que está sendo demonstrada foi realizada sob a orientação e aprovação do Animal Care and Use Committee University of Texas MD Anderson Cancer Centers Institucional (IACUC). <ol><li> Crescer um número apropriado de células (~ 100 k) em um balão de cultura de tecidos com meio de crescimento adequado. Incubar numa incubadora a 37 ° C com 5% de CO 2 durante o período de cultura de células. </li><li> Tratar as células com tripsina (para separar do balão) e produzir uma solução de 2 milhões de células para cada 25 ul. Adicionar uma quantidade igual de Matrigel (no gelo) e misture bem para preparar a solução de injeção final. Injectar esta solução para a posição desejada na parte traseira do rato e esperar uma quantidade apropriada de tempo para os tumores crescerem até ao tamanho desejado (2-4 semanas para a maioria das células). Antes da exposição RF, BALB-C camundongos nus deve ter tumores sólidos ectópica 0,5-1 cm de diâmetro. </li></ol></li><li><ol><li> Preparar os ratos para ser utilizado (neste caso de BALB-C ratinhos nu) por anaesthetizing los com uma solução de cetamina e xilazina por injecção IP. Enquanto os ratos estão caindo no sono, mantê-los em uma câmara com temperatura controlada a 37 ° C. Serão necessários 20 camundongos no total, todos os tumores de tamanho semelhante de rolamento. Dez ratos será usado em conjunto com e sem AuNPs (sendo este último apenas injeções de PBS), enquanto os restantes 10 ratos serão divididos entre os controles expostos à RF não RF-expostos e ambos os grupos: sem injeções AUNP. </li><li> Uma vez devidamente anestesiados, injetar os AuNPs diretamente no tumor, utilizando uma seringa de 1 cc com uma agulha G 27. A solução AUNP deve estar a uma concentração de 200 mg de Au / L em 0,1 mL de PBS. Após a injeção, use um cotonete cirúrgico para absorver o sangue e limpar o local da injecção com um algodão embebido em álcool. </li><li> Em seguida, montar o mouse para ser tratada na cabeça de recepção do gerador de RF. O rato tem de ser posicionado de modo a que o tumor é o mais próximo da cabeça de transmissão. Proteja as áreas que não estão a ser tratados, bem como a sensibilidadetiva áreas como olhos, ouvidos, mãos e pés, com fita de cobre. Certifique-se de que a fita de cobre é apropriadamente entrar em contato com o plano de terra para que não ocorra o acúmulo de carga. Além disso, a área de exposição tem de ter um espaço de pelo menos 1 cm maior do que a dimensão do local de tratamento desejado. </li><li> Posição IV câmara térmica de modo a que o tumor e a área de tratamento são visíveis. Ligue RF por 5 min. Registre a curva da temperatura resultante. Se a terapia atinge temperaturas superiores a 42 ° C de tratamento de parar imediatamente. </li></ol></li><li><ol><li> Após a exposição RF recuperar os ratos da anestesia em uma câmara quente até que eles são conscientes. </li><li> Repita a experiência com o mesmo camundongos 48 horas mais tarde (etapas 3.3.1 - 3.4.1). </li><li> Após a experiência, a eutanásia dos ratos de acordo com os protocolos e procedimentos institucionais. Registar o peso do tumor. Para a análise histológica corrigir os tumores usando formalina e incorporá-los em parafina. Seções tumorais são normalmente manchado sagacidadeh hematoxilina e eosina e para alvos relevantes para medir a eficácia terapêutica, por exemplo, Ki-67, caspase-3, etc. </li></ol></li></ol>

<ol>
	<li>Gannon, C. 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=Carbon+nanotube-enhanced+thermal+destruction+of+cancer+cells+in+a+noninvasive+radiofrequency+field.">Carbon nanotube-enhanced thermal destruction of cancer cells in a noninvasive radiofrequency field.</a> Cancer. 110, 2654 (2007).</li><li>Curley, S. A., Cherukuri, P., Briggs, K., Patra, C. R., Upton, M., Dolson, E., Mukherjee, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+radiofrequency+field-induced+hyperthermic+cytotoxicity+in+human+cancer+cells+using+cetuximab-targeted+gold+nanoparticles.">Noninvasive radiofrequency field-induced hyperthermic cytotoxicity in human cancer cells using cetuximab-targeted gold nanoparticles.</a> J. Exp. Ther. Oncol. 7, 313 (2008).</li><li>Gannon, C. J., Patra, C. R., Bhattacharya, R., Mukherjee, P., Curley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+gold+nanoparticles+enhance+non-invasive+radiofrequency+thermal+destruction+of+human+gastrointestinal+cancer+cells.">Intracellular gold nanoparticles enhance non-invasive radiofrequency thermal destruction of human gastrointestinal cancer cells.</a> Journal of Nanobiotechnology. 6, 2 (2008).</li><li>Raoof, 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=Stability+of+antibody-conjugated+gold+nanoparticles+in+the+endolysosomal+nanoenvironment:+implications+for+noninvasive+radiofrequency-based+cancer+therapy.">Stability of antibody-conjugated gold nanoparticles in the endolysosomal nanoenvironment: implications for noninvasive radiofrequency-based cancer therapy.</a> Nanomedicine. 8, 1096 (2012).</li><li>Glazer, E. S., Massey, K. L., Zhu, C., Curley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+carcinoma+cells+are+susceptible+to+noninvasive+radio+frequency+fields+after+treatment+with+targeted+gold+nanoparticles.">Pancreatic carcinoma cells are susceptible to noninvasive radio frequency fields after treatment with targeted gold nanoparticles.</a> Surgery. 148, 319 (2010).</li><li>Glazer, E. S., Curley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiofrequency+field-induced+thermal+cytotoxicity+in+cancer+cells+treated+with+fluorescent+nanoparticles.">Radiofrequency field-induced thermal cytotoxicity in cancer cells treated with fluorescent nanoparticles.</a> Cancer. 116, 3285 (2010).</li><li>Glazer, E. S., Curley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+radiofrequency+ablation+of+malignancies+mediated+by+quantum+dots,+gold+nanoparticles+and+carbon+nanotubes.">Non-invasive radiofrequency ablation of malignancies mediated by quantum dots, gold nanoparticles and carbon nanotubes.</a> Therapeutic Delivery. 2, 1325 (2011).</li><li>Corr, S. J., Raoof, M., Mackeyev, Y., Phounsavath, S., Cheney, M. A., Cisneros, B. T., Shur, M., Gozin, M., McNally, P. J., Wilson, L. J., Curley, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Citrate-Capped+Gold+Nanoparticle+Electrophoretic+Heat+Production+in+Response+to+a+Time-Varying+Radiofrequency+Electric-Field.">Citrate-Capped Gold Nanoparticle Electrophoretic Heat Production in Response to a Time-Varying Radiofrequency Electric-Field.</a> J. Phys. Chem. C. 116, 24380 (2012).</li><li>Kruse, D. E., 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+Radio-Frequency+Coupling+Network+for+Heating+of+Citrate-Coated+Gold+Nanoparticles+for+Cancer+Therapy:+Design+and+Analysis.">A Radio-Frequency Coupling Network for Heating of Citrate-Coated Gold Nanoparticles for Cancer Therapy: Design and Analysis.</a> IEEE Transactions on Biomedical Engineering. 58, 10 (2011).</li><li>Li, 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=Negligible+absorption+of+radiofrequency+radiation+by+colloidal+gold+nanoparticles.">Negligible absorption of radiofrequency radiation by colloidal gold nanoparticles.</a> J. Colloid Interf. Sci. 358, 47 (2011).</li><li>Liu, X., Chen, H. J., Chen, X., Parini, C., Wen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+frequency+heating+of+gold+nanoparticle+dispersions+for+non-invasive+thermal+therapies.">Low frequency heating of gold nanoparticle dispersions for non-invasive thermal therapies.</a> Nanoscale. , (2012).</li><li>Sassaroli, E., Li, K. C. P., O'Neill, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radio+frequency+absorption+in+gold+nanoparticle+suspensions:+a+phenomenological+study.">Radio frequency absorption in gold nanoparticle suspensions: a phenomenological study.</a> J. Phys. D App. Phys. 45, 075303 (2012).</li><li>Worle-Knirsch, J. M., Pulskamp, K., Krug, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oops+they+did+it+again!+Carbon+nanotubes+hoax+scientists+in+viability+assays.">Oops they did it again! Carbon nanotubes hoax scientists in viability assays.</a> Nano Lett. 6, 1261 (2006).</li></ol>

Não temos nada a divulgar.

Estes protocolos permitem o experimentalista completamente a analisar a medida em que os nanomateriais (neste caso AuNPs) pode aumentar a hipertermia induzida por RF para o tratamento do cancro. O primeiro protocolo trata especificamente analisando a produção de calor a partir de amostras AUNP altamente concentrada e purificada. Embora outros grupos relataram a produção de calor, principalmente a partir dos tampões que os AuNPs são suspensos e não as próprias 9-11 AuNPs, os seus sistemas de RF utiliza...

A absorção de energia de RF por um tecido biológico (devido à sua permitividade eléctrica inerente) resulta em temperaturas elevadas do tecido em função do tempo, o que eventualmente leva a morte celular por hipertermia. Supõe-se que a hipertermia pode ser optimizado para o cancro através da utilização de nanomateriais segmentados que internalizam dentro da célula cancerosa e actuar como transdutores de RF-térmicos, deixando as células normais vizinhas, saudáveis ​​intactas. Vários relatos já mostraram que uma variedade de NPs podem atuar como fontes de calor RF eficaz que ajuda na necrose câncer 1-4. Nestes aspectos, NPs de ouro (AuNPs) 3-5, os nanotubos de carbono 1, e pontos quânticos 6, 7 exibiram características emocionantes, quando utilizado em estudos in vitro e in vivo em experimentos de RF. Embora a natureza exata do mecanismo de aquecimento dessas NPs quando expostos a um campo de RF ainda está sendo debatido, uma série deexperiências fundamentais usando AuNPs tem colocado grande importância tanto tamanho NP e os estados de agregação. Foi demonstrado que apenas AuNPs com diâmetros &lt;10 nm aquecerá quando expostos a um campo de RF-8. Além disso, este mecanismo de aquecimento é atenuada significativamente quando os AuNPs são agregados. Esta condição agregação também foi validado dentro de modelos in vitro que colocaram importância sobre otimização AUNP estabilidade coloidal dentro de compartimentos intracelulares endolysomal para a terapia eficaz RF 4. No entanto, as técnicas e os princípios experimentais utilizados para coletar e avaliar esses dados pode ser problemático, especialmente no caso de validar perfis calor RF de colóides NP. Vários estudos têm mostrado que o aquecimento Joule da suspensão iônica fundo que os NPs são suspensas pode ser a principal fonte de produção de calor de RF e não os próprios 9-12 PN. Embora o nosso trabalho recente 8 validou tA utilização da RF interações na geração de calor a partir AuNPs de diâmetro inferior a 10 nm, pretendemos descrever esses protocolos com mais detalhes ao longo deste artigo. Nós também demonstramos os protocolos e as técnicas necessárias para avaliar a eficácia de AuNPs como agentes térmicos hipertermia, tanto in vitro e in vivo para os modelos de câncer de fígado. Embora nos concentrar-se principalmente no colóides simples de AuNPs cobertas de citrato, as mesmas técnicas podem ser aplicadas a outros híbridos AUNP tais como complexos anticorpo-e quimioterapia conjugado. Ao aderir a esses princípios o experimentalista deve esperamos ser capazes de avaliar rapidamente o potencial de qualquer nanomaterial para ser um agente hyperthermic térmica induzida pela RF eficaz.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagent/Material</td>
		</tr>
		<tr>
			<td>500 ml gold nanoparticles (5 nm)</td>
			<td>Ted Pella, INC</td>
			<td>15702-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amicon Ultra-4/-15 Centrifugal Filter Units (50 kDa)</td>
			<td>Millipore</td>
			<td>UFC805024/UFC910096</td>
			<td>(4 ml and 15 ml volumes)</td>
		</tr>
		<tr>
			<td>MEM X1 Cell Culture Media</td>
			<td>Cellgro</td>
			<td>10-101-CV</td>
			<td>(add extra nutrients as necessary)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F4135-500 ml</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Copper Tape</td>
			<td>Ted Pella</td>
			<td>16072</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Kanzius RF System (13.56 MHZ)</td>
			<td>ThermMed, LLC, Inc. (Erie, PA, USA)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IR Camera</td>
			<td>FLIR SC 6000, FLIR Systems, Inc. (Boston, MA, USA)</td>
			<td>Contact FLIR</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1.3 ml Quartz Cuvette</td>
			<td>ThermMed, LLC, Inc. (Erie, PA, USA)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Teflon Sample holder with Rotary Stage</td>
			<td>ThermMed, LLC, Inc. (Erie, PA, USA)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SPECTROstar Nano Microplate reader</td>
			<td>BGM Labtech</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>UV-Vis spectrometer</td>
			<td>Applied Nanofluorescence, Houston, TX)</td>
			<td>NS1 NanoSpectralyzer</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ICP-OES</td>
			<td>PerkinElmer</td>
			<td>Optima 4300 DV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Zetasizer</td>
			<td>Malvern</td>
			<td>Zen 3600 Zetasizer</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

protocols for assessing radiofrequency interactions with gold nanoparticles and biological systems for non-invasive hyperthermia cancer therapy

Terapias do cancro, que são menos tóxicos e invasivo do que os seus homólogos existentes são altamente desejáveis. O uso de RF-campos elétricos que penetram profundamente no corpo, causando toxicidade mínima, estão actualmente a ser estudada como um meio viável de tratamento do câncer não-invasivo. Prevê-se que as interações de energia de RF com nanopartículas internalizadas (PN) pode libertar calor que pode causar superaquecimento (hipertermia) da célula, em última análise, que termina em necrose celular. No caso de sistemas não-biológicos, apresentamos protocolos detalhados relativos a quantificação do calor liberado por colóides NP altamente concentrados. Para os sistemas biológicos, no caso de experimentos in vitro, descrevemos as técnicas e as condições que devem ser observadas, a fim de expor eficazmente as células cancerosas à energia de RF, sem mídia artefatos de aquecimento a granel obscurecendo significativamente os dados. Finalmente, damos uma metodologia detalhada fou em modelos in vivo de mouse com tumores de câncer hepático ectópica.

1. Avaliando aquecimento RF de colóides NP: AuNPs como exemplo. Depois de seguir a seção 1.1 - 1.2.3 esperamos ter uma solução estável e purificada altamente concentrado de 5 nm e 10 nm de diâmetro AuNPs. A partir dos 500 ml de solução de estoque-comprados, esperamos obter, pelo menos, 4 ml de solução, a uma concentração de 1,000 mg / L. A diferença de horas entre a AuNPs e a solução tampão de água DI fundo com esta concentração deve ser ~ 0,25 ° C / seg e 0,1 ° C / seg a ...

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Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy | Medicine | Video

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

Descrevemos os protocolos utilizados para investigar as interações de 13,56 MHz de radiofreqüência (RF) elétricos campos com colóides de nanopartículas de ouro em ambos os sistemas não-biológicos e biológicos (in vitro / in vivo). Essas interações estão sendo investigados para aplicações na terapia do câncer.

Protocolos para Avaliação de radiofreqüência Interações com Nanopartículas de Ouro e Sistemas biológicos para terapia não-invasiva Câncer Hipertermia

Cancer therapies  which are less toxic and invasive than their existing counterparts are highly  desirable. The use of RF electric-fields that penetrate ...

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10.7K visualizações.  University of Texas M.D. Anderson Cancer Center.  Descrevemos os protocolos utilizados para investigar as interações de 13,56 MHz de radiofreqüência (RF) elétricos campos com colóides de nanopartículas de ouro em ambos os sistemas não-biológicos e biológicos (in vitro / in vivo). Essas interações estão sendo investigados para aplicações na terapia do câncer. 

Vídeo: Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Protocolos de coloração de ilhotas pancreáticas humanas

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Medicina

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

Luminescência de Cerenkov Imaging (CLI) para o Monitoramento Terapia de Câncer

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Não-invasivo de medição óptica do metabolismo cerebral e Hemodinâmica em Lactentes

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

Avaliação não invasiva da função microvascular e endotelial

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

Transplante para a câmara anterior do olho para longitudinal, não-invasivo<em&gt; Em vivo</em&gt; Imaging com uma única célula de resolução em tempo real

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to return to normal in a large majority of patients2, which is mainly due to current lack of clinical treatment for acute stroke. This necessitates understanding the physiological effects of cerebral ischemia on brain tissue over time and is a major area of active research. Towards this end, experimental progress has been made using rats as a preclinical model for stroke, particularly, using non-invasive methods such as 18F-fluorodeoxyglucose (FDG) coupled with Positron Emission Tomography (PET) imaging3,10,17. Here we present a strategy for inducing cerebral ischemia in rats by middle cerebral artery occlusion (MCAO) that mimics focal cerebral ischemia in humans, and imaging its effects over 24 hr using FDG-PET coupled with X-ray computed tomography (CT) with an Albira PET-CT instrument. A VOI template atlas was subsequently fused to the cerebral rat data to enable a unbiased analysis of the brain and its sub-regions4. In addition, a method for 3D visualization of the FDG-PET-CT time course is presented. In summary, we present a detailed protocol for initiating, quantifying, and visualizing an induced ischemic stroke event in a living Sprague-Dawley rat in three dimensions using FDG-PET.

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with 18F-FDG

Brain damage resulting from cerebral ischemia may be non-invasively imaged and studied in rats using pre-clinical positron emission tomography coupled with the injectable radioactive probe, 18F-fluorodeoxyglucose. Further, the use of modern software tools that include volume of interest (VOI) brain templates dramatically increase the quantitative information gleaned from these studies.

Imagem não invasivo e Análise de Isquemia Cerebral em Ratos de Vida Usando Positron Emission Tomography com<sup&gt; 18</sup&gt; F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Modelo murino de imagem não invasivo para detectar e monitorar o cancro do ovário Recorrência

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Avaliação não invasiva da eficácia de novos Therapeutics para Intestinal Patologias Usando imagem endoscópica série da Vivo Ratos

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Avaliação não-invasiva da função de músculos levantadores em ratos

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Oxygenation-sensitive Cardiac MRI with Vasoactive Breathing Maneuvers for the Non-invasive Assessment of Coronary Microvascular Dysfunction

Oxygenation-sensitive cardiac magnetic resonance imaging (OS-CMR) is a diagnostic technique that uses the inherent paramagnetic properties of deoxyhemoglobin as an endogenous source of tissue contrast. Used in combination with standardized vasoactive breathing maneuvers (hyperventilation and apnea) as a potent non-pharmacologic vasomotor stimulus, OS-CMR can monitor changes in myocardial oxygenation. Quantifying such changes during the cardiac cycle and throughout vasoactive maneuvers can provide markers for coronary macro- and microvascular function and thereby circumvent the need for any extrinsic, intravenous contrast or pharmacologic stress agents.
OS-CMR uses the well-known sensitivity of T2*-weighted images to blood oxygenation. Oxygenation-sensitive images can be acquired on any cardiac MRI scanner using a modified standard clinical steady-state free precession (SSFP) cine sequence, making this technique vendor-agnostic and easily implemented. As a vasoactive breathing maneuver, we apply a 4-min breathing protocol of 120 s of free breathing, 60 s of paced hyperventilation, followed by an expiratory breath-hold of at least 30 s. The regional and global response of myocardial tissue oxygenation to this maneuver can be assessed by tracking the signal intensity change. The change over the initial 30 s of the post-hyperventilation breath-hold, referred to as the breathing-induced myocardial oxygenation reserve (B-MORE) has been studied in healthy people and various pathologies. A detailed protocol for performing oxygen-sensitive CMR scans with vasoactive maneuvers is provided.
As demonstrated in patients with microvascular dysfunction in yet incompletely understood conditions, such as inducible ischemia with no obstructive coronary artery stenosis (INOCA), heart failure with preserved ejection fraction (HFpEF), or microvascular dysfunction after heart transplantation, this approach provides unique, clinically important, and complementary information on coronary vascular function.

The assessment of microvascular function by oxygenation-sensitive cardiac magnetic resonance imaging in combination with vasoactive breathing maneuvers is unique in its ability to assess rapid dynamic changes in myocardial oxygenation in vivo and, thus, may serve as a critically important diagnostic technique for coronary vascular function.