This work was supported by the National Research Foundation (NRF-NRFF2011-01) and the National Medical Research Council (NMRC/CBRG/0005/2012).

1. Extração de esporopolenina exina Cápsulas (segundos) a partir de L. clavatum Esporos Nota: O processo de extração SEC envolve um pó inflamável (L. clavatum), ácidos corrosivos picantes, e solventes inflamáveis, equipamentos, portanto, adequada proteção individual (óculos de proteção, máscara, luvas, jaleco), avaliação de risco aprovada em uso e descarte de produtos químicos por pessoal de laboratório autorizado é essencial. <ol><li> Spore Desengordurante <ol><li> Prepara-se uma set-up refluxo numa câmara exaustora, utilizando um condensador de vidro, sistema de circulação de água, e banho de água (Figura 1). </li><li> Pesar 100 g L. esporos clavatum sem criar poeira e longe de qualquer fonte de ignição. Nota: Os esporos aqui utilizados foram obtidos comercialmente (Veja Lista de Materiais). </li><li> Transferência de esporos lentamente a uma de 1 L de politetrafluoretileno (PTFE) frasco de fundo redondo com uma barra de agitação magnética. </li><li> Adicionar 500 ml de acetona para os esporos dobalão de PTFE e agitar suavemente o frasco para formar uma suspensão homogénea. </li><li> Colocar o balão de PTFE no conjunto do banho de água a 50 ° C e conectar-se a um condensador de refluxo. </li><li> Realizar a refluxo em acetona durante 6 horas com agitação a 200 rpm e, em seguida, deixa-se arrefecer à temperatura ambiente. </li><li> Filtrar a suspensão, com papel de filtro sob vácuo e recolher os esporos desengordurados em grandes (15 cm de diâmetro) placas de petri de vidro. </li><li> Secam-se os esporos à temperatura ambiente (hotte) por revestimento com folha de alumínio com furos para a evaporação do solvente. </li></ol></li><li> acidï¿½ise <ol><li> Prepare a 85% (v / v) de ácido fosfórico com água desionizada (Dl) e transferir os esporos secos desengordurados para um balão de 1 L de PTFE. </li><li> Adicionar 500 ml de ácido fosfórico (85% v / v) para os esporos desengordurados. </li><li> Colocar o balão de PTFE em conjunto um banho de água a 70 ° C e conectar-se a um condensador de refluxo. </li><li> Realizar refluxo suave (70 ° C) em ácido fosfórico durante 30 h e, em seguida, para permitiresfriar em temperatura ambiente. </li><li> Diluir a suspensão utilizando água DI 500 ml quente e recolher os SECs por filtração a vácuo utilizando papel de filtro. </li><li> Transferir a segundos para uma proveta de vidro de 3 L para executar uma série de lavagens. </li></ol></li><li> SEC lavar roupa <ol><li> Colocar a proveta de vidro de 3 L contendo SECs numa hotte. </li><li> Adicionar (50 ° C) de água DI 800 ml quente para os SECs com agitação suave durante 10 min. </li><li> Filtrar a suspensão usando papel de filtro e uma filtração a vácuo set-up. </li><li> Recolher o SECs em 3 L copo de vidro limpo. </li><li> Repita os passos 1.3.1. a 1.3.4. cinco vezes. </li><li> Adicionar acetona 600 ml quente (45 ° C) para os SECs com agitação suave durante 10 min. </li><li> Recolher os SECs por filtração sob vácuo e transferir os segundos para a 3 L copo de vidro limpo. </li><li> Repita os passos 1.3.6. e 1.3.7. utilizando-se 600 ml quente (50 ° C) de ácido clorídrico 2M. </li><li> Repita os passos 1.3.6. e 1.3.7. utilizando 600 ml hot (50 ° C) a 2 M de hidróxido de sódio. </li><li> Repita os passos 1.3.1 e 1.3.4 oito vezes. </li><li> Repita os passos 1.3.6. e 1.3.7. utilizando-se 600 ml quente (45 ° C) de acetona. </li><li> Repita os passos 1.3.6. e 1.3.7. utilizando-se 600 ml quente (45 ° C) de etanol. </li><li> Repita os passos 1.3.6. e 1.3.7. utilizando 800 ml quente (50 ° C) de água DI. </li><li> Transferir os SECs limpas para seis grandes placas de petri de vidro e seco numa hote à temperatura ambiente durante 24 horas para remover todo o conteúdo de água. </li><li> Secam-se as SECs num forno de vácuo a 60 ° C e 1 mbar de vácuo durante 10 h ou até peso constante. </li><li> Recolher o SECs seco e transferir para um frasco de polipropileno. </li><li> Armazenar o SECs em um armário seco. </li></ol></li></ol> 2. Caracterização do SECs <ol><li> Realizar electrónica de varrimento análise microscópica utilizando 41 esporos e SECs produzidas em diferentes fases. </li><li> Realizar análise elementar 41 usando esporos e SECs produzidas em diferentes fases. DRY todas as amostras a 60 ° C durante pelo menos 1 hora antes da análise elementar e calcular o teor de proteína usando por cento de azoto, com um factor de multiplicação de 6,25. 11 obter resultados utilizando medições triplicadas para cada amostra. </li><li> Realizar uma análise de propriedades micromerítica usando um analisador de imagem de partículas dinâmico. 41 </li><li> Digitalizar não transformados, processados e SECs FITC-BSA-carregados usando microscopia confocal de varredura a laser. 41 </li></ol> 3. Biomacromolecule Encapsulation por vácuo Carregando Técnica <ol><li> Preparar 1,2 ml solução de 125 mg / ml de albumina de soro bovino (BSA) usando água Dl e transferir para um tubo de polipropileno de 50 ml. </li><li> Pesar 150 mg de segundos e transferir para a solução de BSA. </li><li> Vortex o tubo durante 10 minutos para formar uma solução homogénea. </li><li> Cobrir o tubo com papel de filtro. </li><li> Transferir o tubo para um frasco de secador por congelação e seco durante 4 horas com vácuo a 1 mbar. </li><li> Execute as etapas de 3.1para 3,5. para três lotes independentes. </li><li> Recolher o SECs BSA-carregado e remover os aglomerados usando um almofariz e pilão. </li><li> Transfira os SECs BSA-carregados para um tubo de 50 ml e adicionar 2 ml de água DI para remover a BSA residual. </li><li> Recolher os SECs por centrifugação a 4704 xg durante 5 min e desprezar o sobrenadante. </li><li> Repetir a lavagem com água DI vez. </li><li> Cubra o tubo contendo SECs BSA-carregado usando papel de filtro. </li><li> Transferir os segundos para um congelador (-20 ° C) e congelar durante 1 h. </li><li> Congelar secar o SECs, durante 24 horas e armazenar em um congelador até posterior caracterização. </li><li> Prepare os SECs placebo sem BSA utilizando o mesmo processo que nos passos 3.1. a 3.13. </li><li> Prepare os FITC-BSA SECs carregados usando o mesmo processo que nos passos 3.1. a 3.13. </li></ol> 4. Determinação da eficiência de encapsulação <ol><li> Pesar 5 mg de SECs BSA-carregados em 2 ml de tubos de polipropileno. </li><li> Adicionar 1,4 ml de fosfate solução salina tamponada com pH 7,4 (PBS) e agitar com vortex durante 5 min. </li><li> Sonda sonicar a suspensão a 40% de amplitude durante 3 ciclos de 10 seg. </li><li> Filtrar a solução através de um filtro de 0,45 um de polietersulfona (PES). </li><li> Realizar o mesmo procedimento tal como nos passos 4.1. para 4,4. usando SECs placebo. </li><li> Medir a absorvância a 280 nm, utilizando placebo como um branco. </li><li> Quantificar a quantidade de BSA encapsulada utilizando uma curva padrão de BSA (200 a 1000 ug / ml) em PBS 42. </li></ol>

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Mater. 190 (1), 324-329 (2011).</li><li>Carr, J., Zhang, L., Davis, M., Ravishankar, S., Flieg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale+Controlling+Chemical+Additives+for+Phosphoric+Acid+Production+Plants.">Scale Controlling Chemical Additives for Phosphoric Acid Production Plants.</a> Procedia Engineering. 83, 233-242 (2014).</li><li>Mundargi, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eco-friendly+streamlined+process+for+sporopollenin+exine+capsule+extraction.">Eco-friendly streamlined process for sporopollenin exine capsule extraction.</a> Sci. Rep. 6, 1-14 (2016).</li><li>Smith, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fundamentals of Fourier transform infrared spectroscopy. , 182 (2011).</li></ol>

Os autores não têm nada para revelar.

Neste trabalho, uma análise sistemática da extração SEC de L. esporos clavatum é apresentada e este relatório mostra que é possível produzir cápsulas de qualidade superiores, ao mesmo tempo alcançar uma simplificação significativa do pré-existente protocolo vulgarmente usado. 11 Em contraste com o protocolo já existente que requer uma temperatura elevada de processo (180 ° C) e uma longa duração do processo (7 dias), a corrente 11 SEC optimização processamento e...

Existe um interesse significativo em cápsulas à base de plantas naturais, obtidos a partir de esporos de plantas e pólens para uso em aplicações de microencapsulação. 15/01 Na natureza, esporos e pólen proporcionar uma protecção para os materiais genéticos sensíveis contra condições ambientais adversas. A estrutura básica de esporos de plantas e do pólen compreende, tipicamente, uma camada de invólucro exterior (exina), uma camada de invólucro interior (intina), e o material citoplasmático interno. A exina é composta de um biopolímero quimicamente robusto 1,9,10,13,16 referido como esporopolenina eo intina é composta principalmente de materiais celulósicos. 16-18 cápsulas vazias podem ser isoladas por vários processos para a remoção de material de 7,9 citoplasmática , proteínas, e a camada intine. 2,12,16 Estas cápsulas exinas esporopolenina (segundos) fornecer uma alternativa atraente para encapsulantes sintéticos devido à sua distribuição de tamanho estreita e morfologia uniforme. 7,9,13,19,20 adesenvolvimento de processos padronizados para a obtenção de s a partir de várias espécies de plantas, tais como Licopódio clavatum, abre a possibilidade de uma vasta gama de aplicações de microencapsulação nos campos de entrega de drogas, alimentos, cosméticos e 6,10-13,21. A fim de obter SECs, investigadores trataram primeiro os esporos e pólen com solventes orgânicos e submetida a refluxo em soluções alcalinas para remover o conteúdo citoplasmático. 22-25 No entanto, a estrutura da cápsula remanescente foi determinada para conter ainda o intina camada celulósica. A fim de eliminar isto, os investigadores exploraram a utilização do tratamento de hidrólise ácida prolongada utilizando ácido clorídrico, ácido sulfúrico quente, ou ácido fosfórico quente ao longo de vários dias, 22-25 com ácido fosfórico a tornar-se o método preferido de remoção intina SEC. 2 No entanto, em curso investigação ao longo dos anos tem mostrado que vários esporos e pólenes têm diferentes graus de resistência à dura me processarthods comumente usados. 26,27 Alguns esporos e pólenes são completamente degradada e perder toda a integridade estrutural em soluções alcalinas fortes, ou tornar-se fortemente danificada em soluções ácidas fortes. 16 A variabilidade na resposta SEC para condições de tratamento é devido a variações sutis na química estrutura e morfologia do material de exina esporopolenina exina entre espécies. 28 Devido à variabilidade na robustez de cápsulas exinas esporopolenina (segundos), é necessário optimizar as condições de processamento para cada espécie de esporos e pólen. Esporos de plantas da espécie L. clavatum tornaram-se a única fonte mais amplamente estudado da SCEE. Propõe-se que isto é principalmente devido à sua ampla disponibilidade, baixo custo, monodispersity, robustez e química 9,29 Os esporos podem ser facilmente colhidas e conter conteúdo sporoplasmic sob a forma de agrupamentos de 1 -. 2 uM organelos celulares e biomolecules. 11 L. clavatum esporos foram usados como um lubrificante em pó natural, 30,31 base para cosméticos, 30 e 32-36 em fitoterapia para uma ampla gama de aplicações terapêuticas. O SCEE obtidos a partir de L. clavatum foram mostrados para ser mais resistente ao processamento de SECs de outras espécies de esporos e pólen. 2 Após o processamento, as SECs resultantes foram mostrados para manter suas estruturas microridge intrincados e alta uniformidade morfológica, proporcionando uma grande cavidade interna para encapsulamento. 7 estudos indicam que o L. SECs clavatum podem ser utilizados para a encapsulação de fármacos, vacinas, 10,13 11 proteínas, células, 8 7,14 óleos, 5-7,9 e suplementos alimentares. 5,15 observadas eficiências de carregamento SEC são relativamente elevadas em comparação com convencional materiais de encapsulamento. 7 Há também um certo número de benefícios relatadosSEC para encapsulação, tais como a capacidade de mascarar o gosto, 6,10 e para proporcionar um certo grau de protecção natural contra a oxidação. 12 Nos estudos existentes, o método de extracção mais utilizada SEC para L. clavatum baseia-se em quatro etapas principais. O primeiro passo é de refluxo do solvente em acetona durante até 12 h a 50 ° C para secar os esporos. 11 O segundo passo é a refluxo alcalina em hidróxido de potássio a 6% durante até 12 h a 120 ° C para remover citoplasmática e materiais proteicos. 11 Passo três é a refluxo ácido em ácido fosfórico a 85% durante 7 dias a 180 ° C para remover o material intina celulósico. 11 Passo quatro é um processo de lavagem exaustiva utilizando água, solventes, ácidos e bases para remover todo o material não-exina restante e resíduos químicos. Os principais objetivos da extração SEC em relação a aplicações de encapsulamento são para produzir cápsulas que estão vazios de material citoplasmático, livre from proteínas potencialmente alergênicas, e morfologicamente intactos. 2,37 No entanto, do ponto de vista de produção industrial, também é importante considerar factores económicos e ambientais adicionais, tais como, a eficiência energética, a duração de produção, segurança e resíduos resultantes. No que se refere à eficiência de energia, ambas as altas temperaturas e tempos de processamento longos afecta os custos de produção, bem como o impacto ambiental. duração produção e tempo de resposta são factores-chave que influenciam a rentabilidade de processamento. Particularmente preocupante é que o processamento de ácido fosfórico de alta temperatura aumenta a questões de segurança e é conhecido por resultar em escamação corrosivo que leva a aumentos significativos na manutenção da infra-estrutura e atrasos em tempos de rotação lote 38-40. Sempre que possível, minimizando o número de passos necessários pode levar a redução significativa dos resíduos produzidos. No entanto, a normalmente utilizada processo de quatro passos de L. extração clavatum SEC tem simplesmente evolved de décadas de pesquisa e teve pouco otimização processo real. Recentemente, Mundargi et al., 41 fez uma contribuição significativa para os trabalhos em curso neste domínio, através da avaliação e otimização de uma das técnicas de extração SEC mais comumente reportados de forma sistemática. Na primeira secção deste trabalho: desengorduramento de esporos é demonstrado utilizando processamento de acetona a 50 ° C durante 6 horas; sporoplasm procedimentos de remoção e intina são demonstradas utilizando processamento de ácido fosfórico a 85%, a 70 ° C durante 30 h; lavagem extensiva com água, solventes, ácido, e a base é utilizada para demonstrar a remoção de conteúdos sporoplasmic residuais; e SEC secagem é demonstrada utilizando secagem por convecção e vácuo secagem em estufa. Na terceira secção, SEC carregamento vácuo é demonstrada utilizando o carregamento de vácuo de um modelo de proteína, albumina de soro bovino (BSA), seguido de BSA-carregado-SEC lavagem e liofilização. Na quarta seção, a determinção da eficiência de encapsulamento de BSA é demonstrada utilizando centrifugação, a sonda de ultra-sons, e espectrometria de UV / Vis.

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			<td height="21" style="height:21px;width:319px;">Lycopodium clavatum spores (s-type)</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">19108-500G-F</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Bovine serum albumin</td>
			<td style="width:235px;">Sigma</td>
			<td>A2153-50G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">FITC-conjugated BSA</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">A9771-250MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Phosphoric acid (85 % w/v)</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">438081-2.5L&#160;</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Hydrochloric acid</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">V800202&#160;</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Sodium hydroxide</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">S5881-1KG&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Acetone</td>
			<td style="width:235px;">Sigma</td>
			<td style="width:171px;">V800022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Ethanol</td>
			<td style="width:235px;">AcME&#160;</td>
			<td style="width:171px;">C000356</td>
			<td></td>
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			<td height="21" style="height:21px;width:319px;">Deionized water</td>
			<td style="width:235px;">Millipore purified water&#160;</td>
			<td style="width:171px;"></td>
			<td></td>
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			<td height="42" style="height:42px;width:319px;">Qualitative filter paper (grade No. 1, cotton cellulose)</td>
			<td style="width:235px;"></td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Polystyrene microspheres (50 &#177; 1 &#181;m)&#160;</td>
			<td style="width:235px;">Thermoscientific (CA, USA)</td>
			<td style="width:171px;">4250A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Vectashield&#160;</td>
			<td style="width:235px;">Vector labs (CA, USA)</td>
			<td style="width:171px;">H-1000</td>
			<td></td>
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			<td height="63" style="height:63px;width:319px;">Sticky-slides, D 263 M Schott glass, No.1.5H (170 &#956;m, 25 mm x 75 mm) unsterile glass slide</td>
			<td style="width:235px;">Ibidi GmbH (Munich, Germany)</td>
			<td style="width:171px;">10812</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Commercial Lycopodium SECs (L-type)</td>
			<td style="width:235px;">Polysciences, Inc. (PA, USA)</td>
			<td style="width:171px;">16867-1</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Heating plates</td>
			<td style="width:235px;">IKA, Germany</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Scanning electron microscope</td>
			<td style="width:235px;">Jeol, Japan</td>
			<td style="width:171px;">&#160;JFC-1600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Elemental analyzer&#160;</td>
			<td style="width:235px;">Elementar, Germany</td>
			<td style="width:171px;">VarioEL III</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">FlowCam: The benchtop system</td>
			<td style="width:235px;">Fluid Imaging Technologies, USA</td>
			<td>FlowCamVS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Confocal laser scanning microscope</td>
			<td style="width:235px;">Carl Zeiss, Germany</td>
			<td style="width:171px;">LSM710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Freeze dryer</td>
			<td>Labconco, USA&#160;</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">UV Spectrometer</td>
			<td>Boeco, Germany</td>
			<td style="width:171px;">S220</td>
			<td></td>
		</tr>
	</tbody>
</table>

extraction of plant-based capsules for microencapsulation applications

Microcápsulas derivados de esporos ou pólen à base de plantas fornecem uma plataforma robusta para uma gama diversificada de aplicações de microencapsulação. cápsulas esporopolenina exinas (segs) são obtidos quando os esporos ou pólen são processados ​​de modo a remover o conteúdo sporoplasmic internos. As microcápsulas ocas resultantes exibem um elevado grau de uniformidade micromerítica e reter características microestruturais intrincados relacionados com as espécies de plantas particulares. Aqui, demonstramos um processo simplificado para a produção de s a partir de esporos clavatum Lycopodium e para o carregamento de compostos hidrofílicos para estes segundos. O actual procedimento de isolamento SEC foi optimizado recentemente para reduzir significativamente os requisitos de processamento que são convencionalmente utilizados no isolamento SEC, e para assegurar a produção de microcápsulas intactas. Natural L. clavatum esporos são desengordurado com acetona, tratou-se com ácido fosfórico, e extensivamente lavadas para remover sporoplasmiconteúdo c. Depois de desengorduramento acetona, um único passo de processamento usando 85% de ácido fosfórico tem sido demonstrado para remover todo o conteúdo sporoplasmic. Ao limitar o tempo de processamento ácido para 30 horas, é possível isolar SECs limpas e evitar SEC fractura, o que tem sido demonstrado que ocorre com o tempo de processamento prolongado. lavagem extensiva com água, ácidos diluídos, diluir bases e solventes garante que todos os materiais e produtos químicos resíduos sporoplasmic são adequadamente removidos. A técnica de carga de vácuo é utilizada para carregar uma proteína modelo (Albumina de Soro Bovino) como um composto representativo hidrofílico. carregamento de vácuo proporciona uma técnica simples para carregar vários compostos, sem a necessidade de solventes agressivos ou produtos químicos indesejáveis ​​que são frequentemente necessários em outros protocolos de microencapsulação. Com base nestes protocolos de isolamento e de carregamento, segs proporcionar um material promissor para uso em uma grande variedade de aplicações de microencapsulação, tais como, agentes terapêuticos, alimentos, cosméticos, de cuidados pessoais e proddutos.

Processo de Extracção simplificado para esporopolenina exina Cápsulas O L. extracção clavatum SEC foi conseguida por três etapas principais: (1) Desengordurante usando acetona; (2) acidólise com ácido fosfórico a 85% (v / v); e (3) lavagem SEC extensa utilização de solventes. O fluxo do processo de extracção SEC simplificado é apresentado na Figura 1 A -. I Resu...

Watch this Scientific Journal Video about Extraction of Plant-based Capsules for Microencapsulation Applications at JoVE.com

Extraction of Plant-based Capsules for Microencapsulation Applications

Este protocolo / manuscrito descreve um processo simplificado para a produção de cápsulas exinas esporopolenina (segundos) a partir de esporos clavatum Lycopodium e para o carregamento de compostos hidrofílicos para estes segundos.

Cápsulas à base de plantas de extração de para aplicações de microencapsulação

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

extraction-of-plant-based-capsules-for-microencapsulation-applications

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JoVE Journal

Bioengineering

11.9K visualizações.  Nanyang Technological University.  Este protocolo / manuscrito descreve um processo simplificado para a produção de cápsulas exinas esporopolenina (segundos) a partir de esporos clavatum Lycopodium e para o carregamento de compostos hidrofílicos para estes segundos. 

Vídeo: Extraction of Plant-based Capsules for Microencapsulation Applications

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Preparação de fibrina Andaimes 3D para aplicações de cultura de células-tronco

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Bioengenharia

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

Wideband Detector óptico de ultra-som para aplicações Medical Imaging

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

Projetando proteína de seda-seda liga Materiais para aplicações biomédicas

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

In situ TEM de Assembleias Biológicas em Líquido

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Nanopartículas Lipídicas Sólidas (NLS) para intracelular Segmentação Aplicações

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Fabricação de super-hidrofóbicas Materiais Poliméricos para aplicações biomédicas

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Fabricação de Mecanicamente Tunable e Bioativos metal andaimes para aplicações biomédicas

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formação Light-mediada e padronização de hidrogéis para aplicações de cultura celular

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Rápido Mix Preparação de Bioinspirada Nanoscale hidroxiapatita para aplicações biomédicas

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Dois métodos para Decellularization de tecidos vegetais para aplicações de engenharia de tecidos

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...