The authors would like to acknowledge the technical assistance of Alfred Gunasekaran in electron microscopy studies at the Institute of Micromanufacturing at Louisiana Tech University, and Dr. Jim McNamara for assistance with additional microscopy studies. The work described was supported in part by Louisiana board of Regents PKSFI Contract No. LEQSF (2007-12)-ENH-PKSFI-PRS-04 and the James E. Wyche III Endowed Professorship from Louisiana Tech University (to M.D.).

1. Planejamento de Experimentos <ol><li> Determinar o volume de nanocompósitos de cobre necessário para a síntese. Nessa base, escolha um número de frascos de pequeno volume (25 cm2), ou frascos maiores, como indicado a seguir na preparação de materiais. </li><li> Para esta síntese, utilizar uma temperatura de 37 ° C incubadora com 5% de CO 2 e, pelo menos, 40% de humidade. Assegure-se que uma tal incubadora está disponível e que não será perturbado repetidamente ao longo do período de síntese (aproximadamente 24 horas). CUIDADO: abertura e fecho repetido da incubadora certamente irá causar flutuações de temperatura que podem resultar na síntese de alteração das estruturas nanocompósito. </li></ol> 2. Preparação de Materiais <ol><li> Prepare todos os materiais frescos antes do início de uma experiência, através da adição de materiais sólidos para solventes direita antes da síntese é para começar. Manter as soluções concentradas de cistina e cobre materiais de partida em líquido por longos vezes bntes a experiência não é recomendado e pode levar a resultados variáveis. Uma vez aberta a partir do fornecedor, continuo materiais secos envolvendo a parte superior do recipiente com Parafilm partida. Nota: O protocolo seguinte é utilizado como um exemplo para a reacção, em 25 cm2 de frasco de cultura de células utilizando 7 ul de cistina, 6643 uL de água estéril, e 350 ul de CNPs. </li><li> Prepare a / ml solução 2 mg de nanopartículas de cobre pesando, pelo menos, 2 mg de CNPs. Usar luvas descartáveis, durante este passo para evitar possível contato de CNPs com a pele. Coloque as nanopartículas em um frasco vazio estéril 16 ml de vidro. <ol><li> Para o frasco contendo CNPs, adicionar água desionizada estéril no volume apropriado para produzir uma solução 2 mg / ml e a solução vortex durante 20 segundos para proporcionar dispersão das nanopartículas, antes do início de síntese (pelo menos 1 ml de volume total é recomendado). Não encha o mais de meio caminho frasco com água, pois isso irá inibir a mistura em vórtice. CNPs will rapidamente se depositar no fundo do frasco e aparece de cor escura (cinza ao preto). </li><li> Sonicar a solução CNP por 17 minutos à temperatura ambiente para proporcionar dispersão máxima de CNPs antes do início da síntese. Verifique periodicamente para se certificar de que CNPs estão misturando devido a sonicação. Após uma sonicação bem sucedida, CNPs permanecem suspensas na solução durante pelo menos 30 min e a solução será de cor escura. </li></ol></li><li> Pesar massa suficiente de cistina para produzir uma solução / ml 72,9 mg para a síntese. Desde cistina não é diretamente solúvel em água, coloque o cistina pesava um recipiente de pesagem antiestática. <ol><li> Para a pesagem recipiente que contém cistina, adicionar um volume suficiente de NaOH 1 M estéril, de modo que a cistina dissolve-se completamente. Por exemplo, dissolve-se 7,29 mg de cistina completamente em 100 ul de NaOH 1 M, para fazer um / ml de solução 72,9 mg. </li><li> Para manter condições estéreis, realizar esta etapa em um tecido fluxo cultura capô estéril. CUIDADO: NaOHem 1 M concentração é cáustico, por isso use luvas descartáveis, durante este passo para evitar o contato da pele com NaOH concentrado </li></ol></li><li> Trabalhando numa cultura de tecidos hote estéril, adicionar 7 ul de cistina com 6643 uL de água estéril para a síntese balão estéril em primeiro lugar, e deixar incubar durante 30 minutos na incubadora a 37 ° C, com a tampa do frasco ventilado (solta) para fornecer eficaz de mistura. Ressuspender o CNP solução de 2 mg / ml em vortex durante 30 segundos, uma vez que terá CNPs liquidados após o passo de sonicação. <ol><li> Adicionar solução CNP no balão de síntese (utilizando uma técnica estéril) para manter as proporções dos componentes seguintes: combinar uma partes cistina, 50 partes CNPS, e 949 partes de água estéril num frasco de cultura de célula de 25 2 cm, para iniciar a síntese. Por exemplo, para um volume de 7 ml de síntese, combinar 7 uL da solução de cistina, 350 ul de CNPs, e 6,643 mL de água estéril. Substituir a tampa no frasco de apertar e de modo que é secure. </li><li> Depois de combinar todos os componentes para a síntese, misture delicadamente no frasco agitando 4-5 vezes. Colocar o balão na incubadora de CO 2 e ventilar o frasco, desapertando o tampão de modo que não haverá permuta de gás dentro e para fora do frasco durante a síntese. </li></ol></li><li> Permitir síntese para executar na incubadora por aproximadamente 24 horas. Durante a síntese, pode-se observar, com microscopia e por olho, formação de compósitos altamente lineares. Nota: O processo de formação das estruturas pode acontecer de repente no sentido em que as estruturas são inicialmente difícil de detectar, em seguida, aparência procede rapidamente a uma densidade crescente. A formação pode, portanto, ocorrer antes de 24 horas. O processo também pode ser observada a olho nu uma vez que as estruturas se tornam maiores e os seus aumentos de densidade. Embora a geração de estruturas pode ser observado ao longo do tempo sob o microscópio e pelo olho em pontos de tempo posteriores, interrompendo continuamente as condições de síntese e temperatura irá levar a um mauresultados de síntese. </li><li> Terminar a síntese de biocompósitos por firmemente o balão de síntese de nivelamento e de armazenar o recipiente no frigorífico (4 ° C). Estruturas, uma vez gerados, permanecer estável nesta forma durante pelo menos um ano. Rotular o frasco com condições de síntese, incluindo componentes utilizados, a data de síntese, e o tempo de incubação da síntese antes da terminação. </li></ol> 3. Síntese Usando sulfato de cobre <ol><li> Realizar a síntese de auto-montagem, substituindo CNPs com sal sulfato de cobre. Utilizando uma técnica estéril, dissolver, pelo menos, 2 mg de sulfato de cobre em um volume suficiente de água desionizada estéril para fazer uma solução / ml 2 mg. Os cristais de sulfato de cobre ir facilmente em solução a esta concentração, mas vortex do frasco, se necessário, e inspeccionar por olho para assegurar que todos os cristais são dissolvidos. </li><li> Após a preparação do sulfato de cobre, realizar a síntese como descrito anteriormente, mas substituindo CNPs com o sulfato de cobre. Nota: nanocompósitos auto-montadas usando sulfato de cobre como um material de partida, verificou-se ser muito mais consistente na forma final do que para estruturas sintetizadas a partir CNPs. </li><li> Terminar a síntese de biocompósitos sulfato de cobre como para os compósitos CNP (passo 2.6) e armazená-las a longo prazo, a 4 ° C. </li></ol> 4. Caracterização e Manuseio de Biocompósitos Pós-síntese <ol><li> Caracterizar biocompósitos derivados de CNPs e de sulfato de cobre por microscopia de luz branca 9 e por microscopia eletrônica de 9. <ol><li> Para a caracterização e inspecção de biocompósitos pós-síntese por microscopia de luz branca, usar um microscópio invertido, como compósitos vai assentar a superfície inferior do balão dentro de poucos minutos após a postura do balão plana, e, em seguida, pode ser trazida para o foco. Utilize a definição de campo claro no microscópio para maximizar o contraste entre biocompósitos e o meio líquido. Composites derivados de CNPs e policialpor sulfato serão ambos parece claro a opaco na cor, mas agregados CNP não reagiram aparece muito escuro na cor. <ol><li> Use uma câmera digital conectada ao microscópio para capturar imagens dos compósitos. Uma gama de comprimentos para as estruturas individuais serão observadas. </li></ol></li><li> Para a caracterização e inspecção de biocompósitos pós-síntese e depois da armazenagem a 4 ° C, para permitir frascos vir até à temperatura ambiente durante pelo menos 15 minutos como tubos vai formar condensação inicialmente após a remoção do frigorífico, o que irá obscurecer eficaz de focagem durante a realização de microscopia de imagem. Depois de permitir o equilíbrio à temperatura ambiente, limpar as superfícies de topo e de fundo do balão com uma toalha de papel limpa para maximizar a qualidade de imagem de microscopia. </li><li> Ao trabalhar com ou de imagem compósitos que foram armazenados a longo prazo, vortex o frasco durante 30 segundos para dissociar aglomerados de compósitos que formam enquanto na geladeira. Depois de vórtex, inspecionar as estruturas com um microfone invertidoroscope para assegurar que os agregados foram dissociadas, e repetir vórtice, conforme necessário. </li><li> Use microscopia de luz branca invertida para avaliar a eficácia da síntese para uma dada experiência utilizando CNPs. Por exemplo, documentar a presença ou ausência de que não reagiu CNPs em frascos de síntese utilizados para biocompósitos CNP-derivadas a partir de frascos com diferentes parâmetros tais como o tempo de síntese. Nota: Pessoa CNPs são demasiado pequenos para observar com um microscópio de luz, mas que não reagiu CNP agregados aparece como-forma redonda e objectos escuras, em contraste com os CNP-compósitos sintetizados com sucesso, que terá uma relação de aspecto elevada, forma linear, e terá uma gama de diferentes comprimentos. Evitar a realização de síntese para demasiado longo de um período de tempo antes da rescisão, como isso irá resultar em muito ramificados estruturas do tipo &quot;urchin&quot;, que são difíceis de dispersar em estruturas individuais, uma vez formados. </li><li> Use microscopia de luz branca invertida para avaliar a eficáciada síntese para um dado experimento usando sulfato de cobre. Desde sulfato de cobre vai plenamente em solução usando este protocolo, a solução aparece menos escura do que a solução de síntese usando CNPs. Documentar o tamanho ea extensão de compósitos de sulfato de cobre, comparando frascos com diferentes condições de síntese, tais como o tempo de síntese antes da rescisão. Nota: compostos sintetizados com sucesso vai mostrar uma gama de diferentes comprimentos. Evitar a realização de síntese para demasiado longo de um período de tempo antes da rescisão, como isso irá resultar em agregados altamente ramificados de compósitos, alguns dos quais serão &quot;ouriço-like&quot; na estrutura, e que são difíceis de dispersar em estruturas individuais, uma vez formados . </li></ol></li><li> Para concentrar biocompósitos pós-síntese, as soluções de compostos de centrifugação num tubo de centrífuga. Adicionar 6 mL quer de estruturas derivadas do CNP ou estruturas derivadas de sulfato de cobre para um tubo de centrífuga de 15 ml. Centrifugar durante 10 mina 500 xg à temperatura ambiente para formar um pelete. Para volumes menores, adicione 500 mL de estruturas em solução a 0,6 ml tubos porte. Centrifugar a 2000 xg à temperatura ambiente durante pelo menos 10 minutos para formar uma pelete. <ol><li> Após centrifugação durante tempo suficiente (pelo menos 10 min para microfuges), guardar o sedimento observável na parte inferior do tubo, onde as estruturas são concentrados por remoção cuidadosa do líquido sobrenadante acima da pelete. Estruturas biocomposite derivados de sulfato de cobre aparecem na cor azul e estruturas derivadas CNPs são mais escuras (cinza ao preto). </li><li> Adicionar mais compósitos para este tubo e repetir o processo no mesmo tubo para concentrar estruturas se desejado. Para dispersar os peletes concentradas, adicionar o volume desejado de solução para o tubo, e vortex durante 10-30 seg. </li></ol></li><li> Sonicar estruturas, uma vez formada, para mover o tamanho da população média (comprimento) das estruturas para valores mais baixos. Estruturas coloque em água deionizada estéril e sonicate por pelo least 10 min. Usando este processo, ao longo do tempo, as estruturas tornam-se fragmentado e menor no comprimento médio (ver figura 6 do texto). Alterações do documento em tamanhos compósitos com diferentes tempos de sonicação utilizando um microscópio invertido luz branca e câmera digital. </li></ol>

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Authors have nothing to disclose.

Ao avaliar os potenciais efeitos tóxicos de nanomateriais incluindo CNPs, observou-se que, a longo prazo, CNPs foram transformados a partir de uma distribuição de partículas mais inicialmente dispersa para uma forma maior, agregados (Figura 2). Em alguns casos, essas formações altamente agregados que foram produzidos na placa de cultura de células, sob condições biológicas, formado projeções altamente linear a partir do centro de agregado, que relembram a cobre anteriormente descrito contend...

Copper is a highly reactive metal that in the biological world is essential in some enzyme functions 1,2, but in higher concentrations is potently toxic including in the nanoparticulate form 3,4. Concern over copper toxicity has become more relevant as CNPs and other copper-based nanomaterials are utilized, due to the increased surface area/mass for nanostructures. Thus, even a small mass of copper, in nanoparticle form, could cause local toxicity due to its ability to penetrate the cell and be broken down into reactive forms. Some biological species can complex with and chelate metal ions, and even incorporate them into biological structures as has been described in marine mussels 5. In studying the potential toxic effects of nanomaterials 4, it was discovered that over time, and under biological conditions used for typical cell culturing (37 &#176;C and 5% CO2), stable copper biocomposites could be formed with a high-aspect ratio (linear) structure.
By a process of elimination, the initial discovery of these linear biocomposites, which occurred in complete cell culture media, was simplified to a defined protocol of essential elements needed for the biocomposites to self-assemble. Self-assembly of two types of highly linear biocomposites was discovered to be possible with two starting metal components: 1) CNPs and 2) copper sulfate, with the common biological component being cystine. Although more complex, so called &#8220;urchin&#8221; and &#8220;nanoflower&#8221; type copper-containing structures with nanoscale and microscale features have been previously reported, these were produced under non-biological conditions, such as temperatures of 100 &#176;C or greater 6-8. To our knowledge, synthesis of individual, linear copper-containing nanostructures that are scalable in liquid phase under biological conditions has not been previously described. 
One of the starting materials utilized for synthesis of nanocomposites, namely CNPs, has been reported previously to be very toxic to cells 4. It has recently been reported that after the nanocomposites are formed, these structures are less toxic on a per mass basis than the starting NPs 9. Thus, the synthesis described here may be derived from a biological and biochemical reaction that has utility in stabilizing reactive copper species, both in the sense of transforming the NP form into larger structures and in producing composites less toxic to cells. 
In contrast to many other nanomaterial forms which are known to aggregate or clump upon interaction with biological liquid media 10,11, once formed, the highly linear composites described here avoid aggregation, possibly due to a redistribution of charge which has been previously reported 9. As detailed in the current work, this avoidance of aggregation is convenient for the purposes of working with the structures once formed for at least 3 reasons: 1) composite structures once formed may be concentrated using centrifugation and then easily dispersed again using vortex mixing; 2) formed structures can be decreased in average size by sonication for different periods of time; and 3) the formed linear structures may provide an additional tool for avoiding the recently described &#8220;coffee ring effect&#8221; 12 and thus provide a dopant for creating more evenly distributed coatings of materials, especially those containing spherical particulates.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mini Vortexer</td>
			<td>VWR (https://us.vwr.com)</td>
			<td>58816-121</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator Model # 2425-2</td>
			<td>VWR (https://us.vwr.com)</td>
			<td>Contact vendor</td>
			<td>Current model calalog # 98000-360</td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge (Refrigerated Microcentrifuge)</td>
			<td>Labnet (http://labnetinternational.com/)</td>
			<td>C2500-R</td>
			<td>Model Prism R</td>
		</tr>
		<tr>
			<td>Cell Culture Centrifuge Model Z323K</td>
			<td>Labnet (http://labnetinternational.com/)</td>
			<td>Contact vendor</td>
			<td>Current model Z206A catalog # C0206-A</td>
		</tr>
		<tr>
			<td>Sonicator (Ultrasonic Cleaner)</td>
			<td>Branson Ultrasonics Corporation (http://www.bransonic.com/)</td>
			<td>1510R-MTH</td>
			<td></td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>Sartorius (http://dataweigh.com)</td>
			<td></td>
			<td>Model CP225D similar model CPA225D</td>
		</tr>
		<tr>
			<td>Olympus IX51 Inverted Light Microscope</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus DP71 microscope digital camera</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>external power supply unit- white light for Olympus microscope</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>TH4-100</td>
			<td></td>
		</tr>
		<tr>
			<td>10x, 20, and 40x microscope objectives</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>Hitachi (http://hitachi-hitec.com/global/em/sem/sem_index.html)</td>
			<td>model S-4800</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>Zeiss (http://zeiss.com/microscopy/en_de/products.html)</td>
			<td>model Libra 120</td>
			<td></td>
		</tr>
		<tr>
			<td>Table Top Work Station Unidirectional Flow Clean Bench</td>
			<td>Envirco (http://envirco-hvac.com)</td>
			<td>model PNG62675</td>
			<td>Used for sterile cell culture technique</td>
		</tr>
	</tbody>
</table>

generation of scalable, metallic high-aspect ratio nanocomposites in a biological liquid medium

O objetivo deste protocolo é descrever a síntese de duas novas biocompósitos com estruturas de relação de aspecto de alta. Os biocompósitos composto de cobre e cistina, quer com nanopartículas de cobre (CNPS) ou sulfato de cobre contribuindo o componente metálico. A síntese é levada a cabo no estado líquido sob condições biológicas (37 ° C) e sob a forma de compósitos auto-montada após 24 h. Uma vez formados, estes compostos são altamente estáveis ​​em ambos os meios líquidos e em uma forma seca. Os compósitos escala nano a partir do micro- gama de comprimento, e de alguns microns a 25 nm de diâmetro. A microscopia electrónica de varrimento com emissão de campo espectroscopia de energia dispersiva de raios-X (EDX), demonstrou que o enxofre estava presente nas estruturas lineares NP-derivados, que não foi encontrada a partir do material de partida CNP, confirmando assim cistina como a fonte de enxofre nos nanocompósitos finais . Durante a síntese destes nano e micro-compósitos lineares, uma variada gama de comprimentos de structures é formada no reactor de síntese. A sonicação da mistura líquida após a síntese foi demonstrado para ajudar a controlar a dimensão média das estruturas, diminuindo a duração média com o aumento do tempo de sonicação. Uma vez que as estruturas formadas são altamente estáveis, não se aglomeram, e são formadas na fase líquida, centrifugação, também podem ser utilizados para auxiliar na concentração e segregar compósitos formados.

A Figura 1 mostra um fluxograma esquemático dos passos de síntese, para formar os biocompósitos lineares descritas neste trabalho. CNPs ou sulfato de cobre como materiais de partida são combinados com água esterilizada para formar uma solução / ml 2 mg, esta solução é misturada e sonicada para fornecer uma mistura uniforme, e esta solução de cobre é, em seguida, misturados na seguinte proporção para a síntese de: 949 partes estéril água: 50 partes de mistura de cobre: ​​1 parte de ...

Watch this Scientific Journal Video about Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium at JoVE.com

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

Aqui apresentamos um protocolo para sintetizar novos, de alta relação de aspecto biocompósitos condições biológicas e em meio líquido. Os biocompósitos escala de nanómetros a micrómetros de diâmetro e de comprimento, respectivamente. Nanopartículas de cobre (CNPS) e sulfato de cobre combinados com a cistina são os componentes chave para a síntese.

Geração de escalável e de alto-Aspect Metallic nanocompósitos índices, em um Biológico Líquido Médio

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

generation-scalable-metallic-high-aspect-ratio-nanocomposites

Research

JoVE Journal

Bioengineering

9.0K visualizações.  Centenary College of Louisiana.  Aqui apresentamos um protocolo para sintetizar novos, de alta relação de aspecto biocompósitos condições biológicas e em meio líquido. Os biocompósitos escala de nanómetros a micrómetros de diâmetro e de comprimento, respectivamente. Nanopartículas de cobre (CNPS) e sulfato de cobre combinados com a cistina são os componentes chave para a síntese. 

Vídeo: Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

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

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

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

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

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

Bioengenharia

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

Teste de microtração ambientalmente controlado mecanicamente adaptativas nanocompósitos poliméricos para<em&gt; Ex vivo</em&gt; Caracterização

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

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 ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

Síntese de nanofibras baseado em queratina de Engenharia Biomédica

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Alternando microgéis de gelatina híbrido Campo-Responsive magnética para controlada de fármacos de libertação

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs &#8212; polysiloxane-based and epoxy-based &#8212; are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 &#176;C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.

We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture.

Preparação de Monodomain cristal líquido elastômeros e de cristal líquido Elastomer nanocompósitos

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Robótico líquido manipulação montagem de dispositivos modulares DNA automática

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Síntese de biocompatíveis cristal líquido de elastómero Espumas como andaimes celulares para cultura de células 3D espaciais

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

Quantificação simultânea de kynureninas selecionadas analisadas por espectrometria de cromatografia líquida-massa em média coletada de culturas celulares cancerosas

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

Geração escalável de organoides cerebelares maduros de células-tronco pluripotentes humanas e caracterização por imunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...