Este trabalho foi financiado pela Fundação Princess Margaret Hospital, o Conselho Nacional de Ciência e do Canadá, o Instituto Canadense de Pesquisa em Saúde, da Fundação Canadense de Inovação Tecnológica em Engenharia, eo Joey e Toby Tanenbaum / Cadeira Bola Brasileiro em Prostate Cancer Research, uma bolsa da Fundação de Pesquisa SUNY e suporte de inicialização da Universidade de Buffalo.

1. Síntese de Pyropheophorbide-lípido <ol><li> Combinar 200 mmol pyropheophorbide (preparado a partir de Spirulina Pacifica algas, tal como descrito anteriormente,... Zheng et al, Bioconj Chem, 2002, 13 -392) 4, 98,7 mg 16:00 lysophophatidylcholine (1-palmitoil-2-hidroxi-sn-glicero -3-fosfocolina, Avanti Polar Lipids # 855675), 76,3 mg de EDC, 48,7 mg de DMAP (4 - (dimetilamino) piridina) em 5 ml de amileno clorofórmio estabilizado por uma proporção de 1:1:2:2 Pyro: Lipid: EDC : DMPA. </li><li> Agita-se a mistura de reacção à temperatura ambiente sob árgon durante 24 horas. Evapora-se o clorofórmio sob pressão reduzida utilizando um evaporador rotativo com aquecimento a 40 ° C. A mistura de isómeros pyro-lípido (piro ligados na posição sn-1 ou sn-2) é produzido. </li><li> Ressuspender mistura isómero piro-lípido em 1 mM de CaCl2, 50 mM de Tris (pH 8), 0,5% de Triton X-100 e 10% de metanol numa concentração de 5 mg / ml. Adicionar fosfolipase A2 (PLA2) de mel de abelha veneno em0,1 mg / ml de concentração e incubar a solução a 37 ° C durante 24 h para digerir sn-1 piro-lípido e para produzir o sn-2 piro-lípido isómero puro. </li><li> Adicionar 2 volumes adicionais de clorofórmio e 1,25 volumes de metanol, extracto, e, em seguida, remover o solvente, utilizando evaporação rotativa a pressão reduzida a 40 ° C. Ressuspender o produto da clivagem em 1% de MeOH em DCM e purifica-se sobre uma pequena coluna de sílica diol para o isomericamente puro piro-lípido. </li></ol> 2. Preparação de Porphysomes <ol><li> Dissolver pyropheophorbide-lipídico, distearoil-sn-glicero-3-fosfoetanolamina-N-metoxi (polietileno glicol) (PEG-2000-PE) e colesterol em clorofórmio separadamente, e gravar as concentrações respectivamente. </li><li> Prepare porphysome filme lipídico em um tubo de ensaio de borosilicato mm de 12x75 com um peso total de lípido de 5 mg / tubo através da combinação de 65% molar de lípido-porfirina, 5% molar de PEG-2000-PE e 30% molar de colesterol. </li><li> Secam-se as películas de lípidos, sob uma corrente de nitrogás gen e deixá-los ainda mais seco sob vácuo por 1 hora. Armazenar as películas lipídicas, a -20 ° C sob atmosfera de árgon até a hidratação e extrusão futuro. </li><li> Adicionar 1 ml de solução salina tamponada com fosfato (PBS, NaCl 150 mM, fosfato 10 mM, pH 7,4) a cada tubo de filme lipídico. Congele o tubo de ensaio em azoto líquido até estar completamente congelada, e em seguida descongelar o tubo em 65 ° C num banho de água, até a amostra ter todos derretido. Vortex o tubo, para um total de 30 seg (3x com 10 seg cada, com 5 seg re-aquecimento a 65 ° C em banho de água entre cada vórtice). </li><li> Repetir o congelamento-descongelamento durante 5 ou mais ciclos de cima do necessário até que não há grandes agregados podem ser observados na solução hidratada. Deixe a suspensão porphysome no gelo quando terminar. </li><li> Montar o extrusor de alta pressão (10 ml) com dois filtros empilhados de 100 nm de policarbonato instalados dentro do extrusor Thermobarrel Ligação a um circulador termostatizado a 65 ° C num banho de circulação de água, e ligar o sistema de extrusão para um tanque de alta pressão por azotoCertifique-se de fornecimento. </li><li> Transferir a suspensão porphysome para a câmara de extrusão usando uma pipeta de transferência de 9 polegadas de vidro. Ajuste a pressão girando o botão no regulador tanque de nitrogênio até uma leitura entre 200 e 300 psig é observado como pressão inicial de extrusão. Abrir a válvula do regulador de pressão gradualmente até caudal porphysome suficiente é alcançado ou um máximo de 800 psig (5440 kPa) de pressão é alcançado. </li><li> Após o primeiro passo de extrusão está completa, fechar a válvula de controlo de pressão e libertar a pressão da extrusora com a abertura da válvula de alívio de pressão na parte superior da extrusora. Reencher a extrusora com a solução porphysome recém-extrudido, e repetir a extrusão de, pelo menos, 10 vezes, para assegurar que a solução final é extrudido homogéneo. </li><li> Determinar a concentração de solução final porphysome medindo a absorção de uma amostra diluída em metanol, por UV-Vis espectrómetro. Calcular a concentração usando a seguinte equação sagacidadeh o coeficiente de extinção de 97000 M -1 cm -1 a 410 nm para pyropheophorbide-lípido. </li></ol><img alt="Equação 1" src="/files/ftp_upload/50536/50536eq1.jpg" /><ol start="10"><li> Meça o tamanho da distribuição final de porphysomes usando um Malvern Zetasizer ZS90 por espalhamento de luz dinâmico. Diluir solução porphysome em PBS e realizar três medições com 15 corridas cada, para a média dos resultados. </li><li> Determinar a extinção da fluorescência de porphysome por comparação da emissão de fluorescência de porphysome temperada (1 mM em PBS) e porphysome extinta (1 mM em 1% de Triton X-100) utilizando um espectrofluorímetro. Tomar os espectros de antes e depois do detergente (Tirton X-100) são adicionados, e a normalizar o sinal de leitura de fluorescência máxima. Defina a excitação de ser 410 nm, e faixa de comprimento de onda de emissão a ser 600-800 nm. </li><li> Manter solução porphysomes sob árgon a 4 ° C até uso futuro, e protegê-la da luz por alumínio fóleo. </li></ol> 3. Preparação de Animais Modelo de Xenoenxerto <ol><li> Cultura das células KB em meio RPMI-1640 (com 10% de FBS), e manter as células em 5% de CO 2 e atmosfera hidratada a 37 ° C. </li><li> Colheita de células KB, usando a técnica de cultura de células padrão. Manter a suspensão de células (20.000 células / ml) em tampão fosfato salino (PBS) em gelo durante o armazenamento a curto prazo até à altura da injecção. </li><li> Prepare um KB xenografts em camundongos nus. Os ratinhos nus são adquiridos em Charles River Laboratories, quando eles são de 20 g (Categoria # CRL: NU (NCr)-Foxn1 nu, 6-7 semanas de idade), dado dieta padrão e de cama (Harlan Laboratories; Dieta: irradiados LM-485, # 7912; cama: cama sabugo, ¼ &quot;, # 7097). Esterilizar todas as ferramentas de manipulação de animais (seringas, fórceps, e almofada animal) antes de experimentos. Anestesiar ratos com isoflurano a 2% v / v em 100% de gás portador de oxigénio. Todos os protocolos de animais são aprovados pela instituição (Universidade Saúde NeTwork neste caso) cuidados com os animais e uso comitê. </li><li> Injectar células tumorais suspensas em PBS subcutaneamente para o flanco direito dos ratinhos. Ratos está pronto para ser utilizado depois de 10 dias, quando tumor atinge diâmetro de 4-5 mm e espessura de 2-3 mm. </li></ol> 4. In vivo Fototérmica Terapia <ol><li> Injetar porphysomes (contendo 750 nmol pyro em volume de 200 mL) por via intravenosa, via veia da cauda. Conduzir a laser irradiação 24 horas pós-injeção de porphysomes. </li><li> Nesta seção, realizar todos os procedimentos em uma sala de segurança do laser fechado. Use óculos de segurança do laser durante a irradiação laser. </li><li> Configure o equipamento de irradiação laser. Segure a fibra de laser e um difusor por uma braçadeira em um carrinho. Defina a potência do laser em nível baixo e ligar o laser. Ajuste a altura da fibra de laser para conseguir uma área de irradiação de 9 mm de diâmetro (DPSS Laser, LaserGlow Technologies, Toronto, Canadá), calibrar a potência do laser de 750 mW (1,18 W / cm 2) com apower metros e, em seguida, desligue o laser para preparar os animais. </li><li> Anestesiar os animais com isoflurano a 2% v / v em 100% gás de transporte de oxigênio pelo nariz cone nenhuma limpeza evidente de isoflurano um perigo risco para a saúde humana). Confirme se o animal é anestesiado profundo o suficiente através da realização de uma pitada do dedo do pé com uma pinça ou os dedos para as patas traseiras do animal. Os animais estão prontos para a irradiação do laser quando eles não respondem a este procedimento. </li><li> Monitorar a temperatura do tumor por uma câmera infravermelha térmica (Mikroshot, LUMASENSE Technologies). Coloque a câmera perto do tumor, focalizá-la bem e ter uma imagem de pré PTT, bem como uma imagem de luz branca normal antes do início da irradiação de PPF. </li><li> Ligue o laser novamente, e aumentar a potência de luz de 750 mW. Posicionar o tumor no centro do feixe de laser para se o tumor completamente coberta, e iniciar a temporização da irradiação. Pegue a imagem temperatura tumor a cada 5 segundos para um tratamento a laser de um minuto. Desligue a laser em 1 min. Stop o fornecimento de gás de 2% de isoflurano, espere até que o animal se recuperar da anestesia e colocá-lo de volta para a jaula. </li><li> Mantenha os ratos em normotermia biotério sob 0:12 hr ciclo claro e escuro de acordo com protocolos de animais aprovados pela University Health Network. Injetar buprenorfina (0,05 mg / kg, por via subcutânea, duas vezes por dia durante uma semana após a irradiação com laser para reduzir a dor causada pela cicatriz. Cada dois dias, a medir o diâmetro do tumor usando uma pinça e tirar uma foto. Calcule o volume do tumor usando o seguinte equação: V = π / 6 · a · b 2, onde a é o diâmetro de comprimento, e b é o curto. </li><li> Sacrificar os animais colocando-os na câmara de CO 2, quando o longo diâmetro do tumor atingir 10 mm, uma vez que é o ponto final definido em nosso laboratório. Realizar luxação cervical após o tratamento com CO2. Traçar a curva de sobrevivência. </li></ol>

<ol>
	<li>Lovell, J. F., 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=Porphysome+nanovesicles+generated+by+porphyrin+bilayers+for+use+as+multimodal+biophotonic+contrast+agents.">Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents.</a> Nat. Mater. 10, 324-332 (2011).</li><li>Lovell, J. F., 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=Enzymatic+regioselection+for+the+synthesis+and+biodegradation+of+porphysome+nanovesicles.">Enzymatic regioselection for the synthesis and biodegradation of porphysome nanovesicles.</a> Angew Chem Int Ed Engl. 51, 2429-2433 (2012).</li><li>Liu, T. W., MacDonald, T. D., Shi, J., Wilson, B. C., Zheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsically+Copper-64-Labeled+Organic+Nanoparticles+as+Radiotracers.">Intrinsically Copper-64-Labeled Organic Nanoparticles as Radiotracers.</a> Angewandte Chemie. , (2012).</li><li>Zheng, G., 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=Low-density+lipoprotein+reconstituted+by+pyropheophorbide+cholesteryl+oleate+as+target-specific+photosensitizer.">Low-density lipoprotein reconstituted by pyropheophorbide cholesteryl oleate as target-specific photosensitizer.</a> Bioconjug Chem. 13, 392-396 (2002).</li><li>Chen, B., Pogue, B. W., Hasan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+delivery+of+photosensitising+agents.">Liposomal delivery of photosensitising agents.</a> Expert Opin Drug Deliv. 2, 477-487 (2005).</li><li>Jin, C. S., Zheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+nanostructures+for+photosensitizer+delivery.">Liposomal nanostructures for photosensitizer delivery.</a> Lasers Surg Med. 43, 734-748 (2011).</li><li>Olson, F., Hunt, C. A., Szoka, F. C., Vail, W. J., Papahadjopoulos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+liposomes+of+defined+size+distribution+by+extrusion+through+polycarbonate+membranes.">Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes.</a> Biochim Biophys Acta. 557, 9-23 (1979).</li><li>Berger, N., Sachse, A., Bender, J., Schubert, R., Brandl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filter+extrusion+of+liposomes+using+different+devices:+comparison+of+liposome+size,+encapsulation+efficiency,+and+process+characteristics.">Filter extrusion of liposomes using different devices: comparison of liposome size, encapsulation efficiency, and process characteristics.</a> Int J Pharm. 223, 55-68 (2001).</li><li>Jesorka, A., Orwar, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes:+technologies+and+analytical+applications.">Liposomes: technologies and analytical applications.</a> Annu Rev Anal Chem (Palo Alto Calif). 1, 801-832 (2008).</li><li>Lapinski, M. M., Castro-Forero, A., Greiner, A. J., Ofoli, R. Y., Blanchard, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+liposomes+formed+by+sonication+and+extrusion:+rotational+and+translational+diffusion+of+an+embedded+chromophore.">Comparison of liposomes formed by sonication and extrusion: rotational and translational diffusion of an embedded chromophore.</a> Langmuir. 23, 11677-11683 (2007).</li><li>Liu, D., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+Homogeneity+of+a+Liposome+Preparation+is+Crucial+for+Liposome+Biodistribution+In+Vivo.">Size Homogeneity of a Liposome Preparation is Crucial for Liposome Biodistribution In Vivo.</a> Journal of Liposome Research. 2, 57-66 (1992).</li><li>Zhang, Y., Lovell, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porphyrins+as+Theranostic+Agents+from+Prehistoric+to+Modern+Times.">Porphyrins as Theranostic Agents from Prehistoric to Modern Times.</a> Theranostics. 2, 905-915 (2012).</li></ol>

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No desenvolvimento de tecnologias de entrega de drogas, nanopartículas multifuncionais são actualmente sob investigação largas para controle preciso do veículo de distribuição de drogas enquanto mantém uma carga de droga elevada. Lipossomas carregados de porfirina têm sido desenvolvidos para obter melhores propriedades farmacocinéticas e de entrega mais eficaz do que a administração directa de porfirina, mas existem obstáculos, incluindo a quantidade de carga restringida de porfirina e redistribuição ráp...

Porphysomes são novos nanovesículas multifuncionais que, recentemente desenvolvidos, que são capazes de imagem e terapia multimodal 1. Eles são formados por camadas duplas porfirina auto-montados e contêm uma altíssima densidade de porfirina (mais de 83, 000/porphysome partículas), o que gera grande coeficiente de extinção e resulta em única dependente de estrutura fluorescência auto-extinção. Porphysomes têm boas in vivo farmacocinéticas e de biodistribuição propriedades: eles exibem uma meia-vida no sangue de 12 horas após a administração sistemática, e passivamente acumulam em tumores de xenoenxerto com 7,5% ID / g às 24 h pós-injecção 2. Sua estrutura única e propriedades físico-químicas fazer porphysome um bom candidato para a imagem latente e terapia multimodal guiada por imagem. Primeiro de tudo, contendo porfirina, porphysomes são intrinsecamente adequado para imagens de fluorescência de tumores após a acumulação do tumor 1.Além disso, cada porfirina tem um local estável de radioisótopos quelantes, por conseguinte, porphysomes pode ser facilmente marcada com radioisótopos, tais como 64Cu para imagiologia PET 3. Além disso, a energia da luz absorvida é dissipada termicamente sob exposição a irradiação com laser quando porphysome estrutura está intacta, assim porphysomes também exibem imagens fotoacústica e capacidades únicas PTT. Demonstrou-se que 24 horas após a injecção intravenosa de porphysomes, irradiação com laser do tumor acumulou-porphysome induziu um rápido aumento de temperatura e de ablação do tumor fototérmica forte. Isto demonstrou que porphysomes são potenciadores fototérmicos eficientes com coeficiente tão alto como as nanopartículas de ouro (AuNPs) extinção 1. Por outro lado, em comparação com outros agentes fototérmicos inorgânicos, incluindo AuNPs, porphysomes mostrar uma vantagem notável em biossegurança, devido à sua natureza orgânica. Porphysomes são enzimaticamente biodegradável e induzir toxicit aguda mínimay em ratos com doses intravenosas tão elevadas como 1000 mg / kg 1. Além disso, semelhante a lipossomas, a grande núcleo aquoso de porphysomes pode ser passiva ou activamente carregados com agentes terapêuticos ou de imagiologia. As propriedades ópticas e de biocompatibilidade porphysomes demonstrar o potencial multimodal de nanopartículas orgânicas para imagiologia biofotônica e terapia. Neste artigo, apresentamos o método de síntese de conjugados pyropheophorbide-lipídico, a fabricação eo método de caracterização de porphysomes utilizando extrusão de alta pressão. In vivo PTT em ratos é realizado, bem como para demonstrar a eficiência da habilitados para porphysome PTT no tumor tratamento utilizando um modelo de tumor de xenoenxerto subcutâneo.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>Thermo-Savant</td>
			<td>SPD131DDA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Votex</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td>Model number: G560</td>
		</tr>
		<tr>
			<td>High pressure extruder</td>
			<td>LIPEX, Northern Lipids Inc.</td>
			<td>T.001</td>
			<td>10 ml Thermobarrel Extruder</td>
		</tr>
		<tr>
			<td>Heated bath circulators (Thermostatted circulator)</td>
			<td>Thermo SCIENTIFIC</td>
			<td>SC100-S5P</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polycarbonate filters</td>
			<td>Avanti Polar Lipids</td>
			<td>610005</td>
			<td>Pore size 100 nm, and membrane diameter 19 mm</td>
		</tr>
		<tr>
			<td>Zetasizer</td>
			<td>Malvern Instruments</td>
			<td>ZS90</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>UV/Visible Spectrophototmeter</td>
			<td>Varian Australia</td>
			<td>Cary 50 Bio UV/ Visible Spectrophotometer</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spectrofluorometer</td>
			<td>HORIBA Scientific</td>
			<td>FluoroMax-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Transmission Electron Microscopy (TEM)</td>
			<td>Hitachi</td>
			<td>H-7000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>SANYO</td>
			<td>MCO-18AIC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Powermeter</td>
			<td>Thorlabs</td>
			<td>PM100D</td>
			<td>with sensor S142C</td>
		</tr>
		<tr>
			<td>671nm Laser</td>
			<td>LaserGlow Technologies</td>
			<td>LRS-0671_PFN-02000-05</td>
			<td>S/N:10097270</td>
		</tr>
		<tr>
			<td>Infrared Thermometer</td>
			<td>Mikroshot, LUMASENSE Technologies</td>
			<td>9102409</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laser protective eye goggles</td>
			<td>LaserGlow Technologies</td>
			<td>AGF6602XX</td>
			<td>Optical Density: 1.5+ at 630-700 nm</td>
		</tr>
	</tbody>
</table>

one minute, sub-one-watt photothermal tumor ablation using porphysomes, intrinsic multifunctional nanovesicles

Recentemente, desenvolveu porphysomes como nanovesículas intrinsecamente multifuncionais. Um fotossensibilizador, pyropheophorbide α, foi conjugado com um fosfolípido e depois auto-montados para vesículas esféricas de lipossomas semelhante. Devido à altíssima densidade de porfirina na bicamada lipídica porfirina, porphysomes gerado grandes coeficientes de extinção, dependente da estrutura de fluorescência auto-extinção, e excelente eficácia fototérmica. Na nossa formulação, porphysomes foram sintetizados utilizando a extrusão de alta pressão, e mostrou um tamanho médio de partícula de cerca de 120 nm. Injecção de vinte e quatro horas após a intravenosa de porphysomes, a temperatura local do tumor aumentou de 30 ° C a 62 ° C rapidamente após a exposição de 750 mW (1,18 W / cm 2), 671 nm, a irradiação com laser de um minuto. Após a ablação térmica completa do tumor, escaras formado e curado dentro de 2 semanas, enquanto que nos grupos de controlo, os tumores continuaram a crescer e todos atingiram o definido brponto d dentro de 3 semanas. Estes dados mostram como porphysomes pode ser utilizado como terapia fototérmica potente (PTT) agentes.

Pyropheophorbide é conjugado com o fosfolípido, como um monómero de lípidos estáveis ​​(Figura 1A) e a conjugados de auto-reunir-se para formar porphysomes pelo método de extrusão de membrana utilizando uma extrusora de alta pressão. Geralmente é difícil para expulsar suspensão porphysome-lipídico durante os 3 primeiros ciclos de extrusão, com taxa de fluxo relativamente lento. Quanto mais as extrusões são repetidos, a taxa de fluxo da solução extrudido aumenta gradualmente, e a pre...

Watch this Scientific Journal Video about One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles at JoVE.com

One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

Nós desenvolvemos novas nanovesículas multifuncionais intrínsecas chamados porphysomes, que têm dependentes de estrutura fluorescência auto-têmpera e propriedades fototérmicos únicas, assim funcionando agentes terapia fototérmica como potentes. Formulamos porphysomes utilizando extrusão de alta pressão e investigou sua eficácia terapia fototérmica em um modelo de tumor xenoenxerto.

One Minute, Sub-One-Watt Photothermal ablação tumoral Usando Porphysomes, Intrínseca Multifuncional nanovesículas

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a ...

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JoVE Journal

Bioengineering

10.7K visualizações.  University of Toronto.  Nós desenvolvemos novas nanovesículas multifuncionais intrínsecas chamados porphysomes, que têm dependentes de estrutura fluorescência auto-têmpera e propriedades fototérmicos únicas, assim funcionando agentes terapia fototérmica como potentes. Formulamos porphysomes utilizando extrusão de alta pressão e investigou sua eficácia terapia fototérmica em um modelo de tumor xenoenxerto. 

Vídeo: One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

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

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

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

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

Bioengenharia

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting airways, whereas adenocarcinomas are typically more peripheral in location. Lung malignancy detection early in the disease process may be difficult due to several limitations: radiological resolution, bronchoscopic limitations in evaluating tissue underlying the airway mucosa and identifying early pathologic changes, and small sample size and/or incomplete sampling in histology biopsies. High resolution imaging modalities, such as optical frequency domain imaging (OFDI), provide non-destructive, large area 3-dimensional views of tissue microstructure to depths approaching 2 mm in real time (Figure 1)2-6. OFDI has been utilized in a variety of applications, including evaluation of coronary artery atherosclerosis6,7 and esophageal intestinal metaplasia and dysplasia6,8-10.
Bronchoscopic OCT/OFDI has been demonstrated as a safe in vivo imaging tool for evaluating the pulmonary airways11-23 (Animation). OCT has been assessed in pulmonary airways16,23 and parenchyma17,22 of animal models and in vivo human airway14,15. OCT imaging of normal airway has demonstrated visualization of airway layering and alveolar attachments, and evaluation of dysplastic lesions has been found useful in distinguishing grades of dysplasia in the bronchial mucosa11,12,20,21. OFDI imaging of bronchial mucosa has been demonstrated in a short bronchial segment (0.8 cm)18. Additionally, volumetric OFDI spanning multiple airway generations in swine and human pulmonary airways in vivo has been described19. Endobronchial OCT/OFDI is typically performed using thin, flexible catheters, which are compatible with standard bronchoscopic access ports. Additionally, OCT and OFDI needle-based probes have recently been developed, which may be used to image regions of the lung beyond the airway wall or pleural surface17.
While OCT/OFDI has been utilized and demonstrated as feasible for in vivo pulmonary imaging, no studies with precisely matched one-to-one OFDI:histology have been performed. Therefore, specific imaging criteria for various pulmonary pathologies have yet to be developed. Histopathological counterparts obtained in vivo consist of only small biopsy fragments, which are difficult to correlate with large OFDI datasets. Additionally, they do not provide the comprehensive histology needed for registration with large volume OFDI. As a result, specific imaging features of pulmonary pathology cannot be developed in the in vivo setting. Precisely matched, one-to-one OFDI and histology correlation is vital to accurately evaluate features seen in OFDI against histology as a gold standard in order to derive specific image interpretation criteria for pulmonary neoplasms and other pulmonary pathologies. Once specific imaging criteria have been developed and validated ex vivo with matched one-to-one histology, the criteria may then be applied to in vivo imaging studies. Here, we present a method for precise, one to one correlation between high resolution optical imaging and histology in ex vivo lung resection specimens. Throughout this manuscript, we describe the techniques used to match OFDI images to histology. However, this method is not specific to OFDI and can be used to obtain histology-registered images for any optical imaging technique. We performed airway centered OFDI with a specialized custom built bronchoscopic 2.4 French (0.8 mm diameter) catheter. Tissue samples were marked with tissue dye, visible in both OFDI and histology. Careful orientation procedures were used to precisely correlate imaging and histological sampling locations. The techniques outlined in this manuscript were used to conduct the first demonstration of volumetric OFDI with precise correlation to tissue-based diagnosis for evaluating pulmonary pathology24. This straightforward, effective technique may be extended to other tissue types to provide precise imaging to histology correlation needed to determine fine imaging features of both normal and diseased tissues.

Optical Frequency Domain Imaging of Ex vivo Pulmonary Resection Specimens: Obtaining One to One Image to Histopathology Correlation

A method to image ex vivo pulmonary resection specimens with optical frequency domain imaging (OFDI) and obtain precise correlation to histology is described, which is essential to developing specific OFDI interpretation criteria for pulmonary pathology. This method is applicable to other tissue types and imaging techniques to obtain precise imaging to histology correlation for accurate image interpretation and assessment. Imaging criteria established with this technique would then be applicable to image assessment in future in vivo studies.

Imagem Domínio óptico Freqüência de<em&gt; Ex vivo</em&gt; Pulmonares espécimes de ressecção: Obtenção de One to One Imagem para Histopatologia Correlação

Lung cancer is the leading cause of cancer-related deaths1. Squamous cell and small cell cancers typically arise in association with the conducting ...

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 ...

Construction of a Preclinical Multimodality Phantom Using Tissue-mimicking Materials for Quality Assurance in Tumor Size Measurement

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for radiologic assessment of solid tumors in response to anti-tumor drug therapy in the 1980s and 1990s, respectively. WHO criteria measure solid tumors in two-dimensions, whereas RECIST measurements use only one-dimension which is considered to be more reproducible 1, 2, 3,4,5. These criteria have been widely used as the only imaging biomarker approved by the United States Food and Drug Administration (FDA) 6. In order to measure tumor response to anti-tumor drugs on images with accuracy, therefore, a robust quality assurance (QA) procedures and corresponding QA phantom are needed.
To address this need, the authors constructed a preclinical multimodality (for ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI)) phantom using tissue-mimicking (TM) materials based on the limited number of target lesions required by RECIST by revising a Gammex US commercial phantom 7. The Appendix in Lee et al. demonstrates the procedures of phantom fabrication 7. In this article, all protocols are introduced in a step-by-step fashion beginning with procedures for preparing the silicone molds for casting tumor-simulating test objects in the phantom, followed by preparation of TM materials for multimodality imaging, and finally construction of the preclinical multimodality QA phantom. The primary purpose of this paper is to provide the protocols to allow anyone interested in independently constructing a phantom for their own projects. QA procedures for tumor size measurement, and RECIST, WHO and volume measurement results of test objects made at multiple institutions using this QA phantom are shown in detail in Lee et al. 8.

This paper describes in-house procedures of constructing a preclinical multimodality phantom made of tissue-mimicking (TM) materials for quality assurance (QA) of tumor size measurement in animal imaging modalities such as ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI).

Construção de um fantasma Multimodalidade pré-clínicos com materiais que imitam tecido para a Garantia da Qualidade no Size Measurement Tumor

World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for ...

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

Produção de grande número de controladas pelo tamanho Spheroids tumorais usando placas de micropoços

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

Medição Longitudinal de Matriz Extracelular Rigidez em 3D tumorais modelos com o uso de rastreamento de partículas Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

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

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

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

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

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

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

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Engenharia de Tecidos por Intrínseca Vascularização numa<em&gt; In Vivo</em&gt; Câmara Engenharia de Tecidos

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis of the intrinsic photothermal response of hemoglobin molecules, the sensor enables high-sensitivity, chemical-free measurement of [Hb]. [Hb] detection capability with a limit of 0.12 g/dl over the range of 0.35 - 17.9 g/dl has been demonstrated previously. The method can be readily implemented using inexpensive consumer electronic devices such as a laser pointer and a webcam. The use of a micro-capillary tube as a blood container also enables the hemoglobin assay with a nanoliter-scale blood volume and a low operating cost. Here, detailed instructions for the PT-AS optical setup and signal processing procedures are presented. Experimental protocols and representative results for blood samples in anemic conditions ([Hb] = 5.3, 7.5, and 9.9 g/dl) are also provided, and the measurements are compared with those from a hematology analyzer. Its simplicity in implementation and operation should enable its wide adoption in clinical laboratories and resource-limited settings.

A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

A rápida e sem produtos químicos dosagem de hemoglobina com Photothermal angular de espalhamento de luz

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis ...