The authors acknowledge financial support from Houston Methodist Research Institute, the National Natural Science Foundation of China (Nos., 21231007 and 21121061), the Ministry of Education of China (Nos., 20100171110013 and 313058), the National Basic Research Program of China (973 Program No. 2014CB845604), and the Fundamental Research Funds for the Central Universities.

1. PCPS Particle Preparação <ol><li> Oxidar partículas de silício poroso não funcionalizados em uma solução a 30% de peróxido de hidrogénio a 95 ° C durante 2 horas. Aminar as partículas oxidadas em 2% (3- aminopropil) trietoxissilano solução em álcool isopropílico durante 2 dias a 65 ° C com agitação suave. </li><li> Centrifuga-se a solução durante 30 min a 18.800 x g e as partículas de lavar duas vezes em álcool isopropílico e três vezes em etanol, utilizando sonicação breve para suspender o sedimento. Deixar as partículas em solução de etanol, quando a execução da etapa 1.3 e 1.4. </li><li> Adicionar um volume conhecido de uma suspensão de partículas (por exemplo, 10 uL) a 10 ml de isótono diluente e contar as partículas com um analisador de contagem de partículas para determinar a concentração de partículas na solução estoque. </li><li> Activa o grupo ácido de L-arginina (0,1 nmol) com a N - (3-dimetilaminopropil) - etilcarbodiimida N &#39;(EDC, 0,1 nmol) / N </em&gt;-Hidroxissuccinimida (NHS, 0,1 nmol) em 20 ml de etanol durante 2 horas à temperatura ambiente com agitação suave. </li><li> Resumidamente sonicar a solução estoque de partículas e adicionar 1 bilhão de partículas à solução de L-arginina e deixar a mistura reaccional durante 18 horas à temperatura ambiente com agitação suave. </li><li> Activa o primeiro grupo ácido aspártico de N - (terc-butoxicarbonil) -L-aspártico (Boc-Asp-OH, 1 nmol) com EDC (0,1 nmol) / NHS (0,1 nmol) em 20 ml de etanol durante 4 horas a 4 ° C com agitação suave. </li><li> Dissolve-se 50 mg de polietilenimina em 10 ml de etanol e adicionar a solução à mistura de Boc-Asp-OH. Deixe que a reacção prosseguir durante 24 horas à temperatura ambiente com agitação suave. </li><li> Activa o segundo grupo ácido aspártico da solução de Boc-Asp-OH / PEI com EDC (0,1 nmol) / NHS (0,1 nmol) a 4 ° C durante 6 horas com agitação suave. </li><li> Para obter partículas de PCPS, adicionar a solução de partículas a partir do passo 1.5 em solução de Boc-Asp-OH / PEI a partir do passo 1.8. Permitir que a reacção prosseguir durante 18 horas à temperatura ambiente com suave r<html> irring. </li><li> Centrifuga-se a solução durante 30 minutos a 18.800 xg e lava-se a solução de partícula três vezes com etanol, utilizando sonicação breve para suspender o sedimento. </li></ol> 2. PCPS Particle Caracterização <ol><li> Medir o tamanho das partículas utilizando um microscópio electrónico de varrimento (SEM). <ol><li> Colocar uma gota de suspensão de partículas (partículas de 10.000 / uL em etanol) em uma amostra de sílica limpa SEM topo e deixar secar à temperatura ambiente sob vácuo. </li><li> Meça MEV a 8 kV com uma distância de trabalho de 3-5 mm, utilizando um detector na lente. </li></ol></li><li> Medir o potencial zeta das partículas usando um sistema de analisador de partículas. <ol><li> Misturar 10 ul de suspensão de partículas (partículas de 10.000 / uL) em etanol com 1 ml de tampão fosfato 10 mM (pH 7,4). </li><li> Carregar a amostra em células capilares dobradas e medir o potencial zeta de acordo com as instruções do fabricante. </li></ol></li></ol>le &quot;&gt; 3. Carregamento de siRNA em PCPS Particles <ol><li> Seque as partículas PCPS (de partícula PCPS preparação passo 1.9) sob vácuo O / N. </li><li> Adicionar siRNA (4 mg) em água livre de nuclease (20 l) para as partículas PCPS secas e brevemente sonicate. Use o seguinte partícula com as taxas de siRNA: 2 × 10 5 partículas / 0,2 ug siRNA, 4 × 10 5 partículas / 0,2 ug siRNA, 6 × 10 5 partículas / 0,2 ug siRNA, 8 × 10 5 partículas / 0,2 mg de siRNA, 10 × 10 5 partículas / 0,2 ug de siRNA e 12 × 10 5 partículas / 0,2 ug de siRNA. </li><li> Incubar durante 3 horas a 4 ° C num agitador (1.000 rpm) para permitir a ligação do siRNA para as partículas. </li></ol> 4. Optimização de siRNA / PCPS Rácio de Partícula <ol><li> Adicione corante DNA para 20 l de partículas siRNA PCPS / controle com partículas diferentes relações siRNA (veja carregamento de siRNA em partículas PCPS). </li><li> Coloque o samples em um gel de agarose 2% contendo mancha gel DNA. </li><li> Efectuar a electroforese a uma voltagem constante de 120 V durante 20 min em tampão de corrida. </li><li> Analisar o gel com aquisição de imagem e software de análise. </li></ol> 5. Lançamento de siRNA de PCPS Particles <ol><li> Misturar 20 ul de partículas de siRNA PCPS / controlo com partículas diferentes relações de siRNA (ver etapa 3) em dodecil sulfato de sódio (SDS, 2%) e deixar repousar durante 1 hora à TA. </li><li> Adicionar corante de carga de ADN para as amostras. </li><li> Amostras de carregar em um gel de agarose 2% contendo mancha gel DNA. </li><li> Execute eletroforese em uma tensão constante de 120 V durante 20 minutos em tampão de corrida usando o equipamento de eletroforese de DNA e uma fonte de alimentação. </li><li> Analisar o gel com aquisição de imagem e software de análise. </li></ol> 6. Microscopia Confocal de PCPS Partículas <ol><li> Adicionar 5 mL de partículas fluorescentes siRNA controle PCPS / (10 × 10 5 partículas / 0.2 mg siRNA / 20 l) para uma lamela de vidro. </li><li> Visualize camadas de partículas por microscopia confocal. </li></ol> 7. Caracterização de Arg-PEI / controle siRNA Nanopartículas <ol><li> Para degradar o material de silício e formar Arg-PEI nanopartículas / siRNA de controlo, adicionar partículas PCPS / siRNA (10 × 10 6 partículas PCPS / 2 ug) siRNA para 100 ul de solução salina tamponada com fosfato e agitar (1000 rpm) a 37 ° C durante 2 dias. </li><li> Centrifuga-se a amostra durante 30 minutos a 18.800 xg e recolher o sobrenadante. </li><li> Medir o tamanho das nanopartículas Arg-PEI / siRNA formados com dispersão de luz dinâmica (DLS). <ol><li> Misturar 10 ul de sobrenadante com tampão de fosfato 1 ml de 10 mM (pH 7,4) e colocar numa cuvete de plástico. </li><li> Medir o tamanho das partículas através de um sistema analisador de partículas de acordo com as instruções do fabricante. </li></ol></li><li> Determinar o tamanho e a morfologia do formado Arg / PEI siRNAnanopartículas com microscopia de força atômica. <ol><li> Tomar 10 ul do sobrenadante e no lugar de uma bolacha de silício. </li><li> Visualize as partículas com microscopia de força atômica (AFM). </li></ol></li></ol> Cultura 8. celular <ol><li> Culturas MDA-MB-231 de cancro da mama humano em meio de cultura celular suplementado com 10% de soro fetal bovino e 1% de penicilina-estreptomicina a 5% de CO 2, 95% de humidade e 37 ° C. </li></ol> 9. microscopia confocal de células vivas com PCPS Particles <ol><li> Células da placa em dois poços de cultura desliza a uma densidade de sementeira de 3 x 10 5 células / poço, durante 24 h. </li><li> Adicionar PCPS partículas carregadas com siRNA controlo fluorescente (10 × 10 5 partículas / 0,2 ug a proporção de partículas de siRNA, 50 nM de ARNsi) para as células. </li><li> Gravar um filme das células com um microscópio confocal (fornecido com uma câmara, 5% de CO2, 95% de humidade e 37 ° C) durante 12 horas seguindo partícula expocerteza. </li></ol> 10. microscopia confocal de células fixadas com PCPS Particles <ol><li> Células da placa em dois poços de cultura desliza a uma densidade de sementeira de 3 x 10 5 células / poço, durante 24 h. </li><li> Adicionar partículas PCPS carregados com siRNA controle fluorescente (10 × 10 5 partículas / 0,2 mg de partículas à relação de siRNA, 50 nM siRNA) para as células e incubar por 1 dia, 7 dias e 10 dias. </li><li> Lavar as células duas vezes com solução salina tamponada com fosfato e depois fixá-los com uma solução de paraformaldeído a 4% durante 10 min. </li><li> Lavam-se as células com solução salina tamponada com fosfato. </li><li> Permeabilizar as células com 0,1% de octil fenol etoxilado durante 10 min e, em seguida, lavar três vezes com solução salina tamponada com fosfato. </li><li> Bloquear as células com albumina de soro de bovino (10 mg / ml) em solução salina tamponada com fosfato durante 10 minutos à temperatura ambiente com agitação suave. </li><li> Para visualizar a actina filamentosa, incubar as células com faloidina marcada fluorescentemente (1 ul / 40 ulsolução) durante 20 min à TA bloqueio com agitação suave e em seguida lavar com solução salina tamponada com fosfato. </li><li> Retirar as lâminas a partir da moldura e adicionar antifade reagente com 4 &#39;, 6-diamidino-2-fenilindole (DAPI) para visualizar o núcleo. </li><li> Adicionar uma tampa de vidro em cima e tomar imagens das células com microscopia confocal. </li></ol> 11. Citometria de Fluxo de células com PCPS / fluorescentes siRNA controle Particles <ol><li> Células da placa em uma placa de 6 poços a uma densidade de sementeira de 3 x 10 5 células / poço, durante 24 h. </li><li> Adicionar PCPS partículas carregadas com siRNA controlo fluorescente (10 × 10 5 partículas / 0,2 ug a proporção de partículas de siRNA, 50 nM de ARNsi) às células e incuba-se durante 24 horas. </li><li> Lavam-se as células com solução salina tamponada com fosfato e raspe-los com um raspador de células. </li><li> Armazenar as células em solução salina tamponada com fosfato com 2% de soro fetal de bovino antes da análise. Utilização de células não tratadas como um controlo negativo. </li><li> Execute cy fluxotometry. </li></ol> 12. A viabilidade celular das células com PCPS Partículas e PCPS / controle siRNA Particles <ol><li> Células da placa em uma placa de 96 poços a uma densidade de células de 3 x 3 10 células / poço, durante 24 h. </li><li> Tratar as células com partículas de PCPS (1,5 x 10 5 / poço e 6 x 10 5 / po) ou partículas de siRNA PCPS / controlo (10 × 10 5 partículas / 0,2 ug a proporção de partículas de siRNA, 10 nM e 100 nM) por 48 siRNA hr e 72 hr. Utilização de células não tratadas e células tratadas com solução salina de fosfato tamponada (mesmo volume que as partículas adicionadas) como controlos. Ensaiar cada amostra em triplicado. </li><li> Realizar um ensaio de proliferação celular de acordo com as instruções do fabricante. </li><li> Representar dados como a média ± desvio padrão. </li></ol> 13. Western Blot de células com PCPS / ATM Mutantes siRNA Particles <ol><li> Células da placa em uma placa de 6 poços, a uma densidade celular de 2 x 10 5 células / well durante 24 h. </li><li> Incubar as células com siRNA PCPS / controle (50 nM) partículas ou PCPS / ATM siRNA (50 Nm) partículas para 72 hr. Utilização de células não tratadas como controle. </li><li> Lisar as células, utilizando um reagente de extracção de proteína suplementado com um cocktail inibidor de protease. </li><li> Centrifuga-se os lisados ​​celulares durante 10 minutos a 14000 xg e recuperar o sobrenadante. </li><li> Determinar a concentração de proteína com um ensaio de quantificação de proteína de acordo com as instruções do fabricante. </li><li> Adicionar tampão de carregamento de amostra (com 5 ul de 2-mercaptoetanol / ml de tampão) para as amostras e aquecê-los durante 6 minutos a 99 ° C. </li><li> Coloque as amostras de proteína (20 ug / ul) num gel de 12% SDS-poliacrilamida em tampão de corrida e efectuar a electroforese em gel de poliacrilamida (1 h, 120 V) usando o equipamento de electroforese e um fonte de alimentação. </li><li> Transferir o gel em tampão de transferência (com 20% de metanol) para uma membrana de nitrocelulose (1 h, 100 V) usando o equipamento de electroforese euma fonte de alimentação. </li><li> Bloquear a membrana com leite em pó a 5% durante 1 hora. </li><li> Incubar a membrana com o anticorpo primário ATM (de coelho) em solução (a partir do passo 13.9) de bloqueio numa concentração de 1: 1000 de diluição O / N. </li><li> Lava-se a membrana com tampão fosfato salino contendo 0,1% de polietileno-glicol-sorbitano e depois incubar com o anticorpo secundário (anti-coelho) em solução (a partir do passo 13.9) de bloqueio a uma diluição de 1: 2500 durante 1 hora. </li><li> Lava-se a membrana com tampão fosfato salino contendo 0,1% de polietileno-glicol-sorbitano e detectar as bandas de proteína com reagente de detecção de Western blot, utilizando software de aquisição de imagem e análise. </li><li> Para o controlo da carga, lava-se a membrana e repetir os passos 13,9-13,12 utilizando um anticorpo primário β-actina (de ratinho, diluição 1: 10.000) e um anticorpo secundário (anti-rato de 1: 4000 de diluição). </li></ol>

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The authors declare that they have no competing financial interests.

Este protocolo descreve um método para a entrega com sucesso e transfecção em células de siRNA. Em particular, a entrega de siRNA é conseguido através de uma plataforma multifuncional que consiste em partículas funcionalizadas psi-policatião. O uso de terapia de siRNA tem grande potencial, por exemplo, o tratamento do cancro, como vários oncogenes pode ser alvo com elevada especificidade. Portanto, existe uma procura para o desenvolvimento de veículos de entrega de siRNA, que pode mitigar os desafios ...

RNAs interferentes (siRNAs) são moléculas de ARN de cadeia dupla que podem suprimir a expressão dos genes. Nos últimos anos, os siRNAs foram desenvolvidos como uma nova geração de biofármacos que mostram um potencial terapêutico para uso futuro em aplicações clínicas 1-5. No entanto, o sucesso da implementação da terapia siRNA continua a ser um desafio considerável, devido à degradação por nucleases, captação intracelular pobres, de baixa eficiência de transfecção e liberação ineficiente do endosome / lisossoma 5. Muitas destas dificuldades podem ser ultrapassadas pelo desenvolvimento de plataformas de entrega, que pode com segurança e eficientemente entregar siRNA para o tecido doente. Em comparação com as transportadoras virais, as plataformas não-virais apresentam várias vantagens, como a segurança, baixo custo e facilidade de alfaiataria. Em particular, as nanopartículas catiónicos, tais como polímeros e lípidos, provaram ser úteis para a entrega de siRNA 3. Anteriormente, temos desenvolvido um dr discoidalug sistema de entrega, o vector denominado de vários estágios (MSV). Esta plataforma é baseada em fases sequenciais, em que um veículo é libertado a partir de uma outra. A primeira fase do veículo é feita a partir de uma micropartícula biodegradável silício poroso (psi), enquanto a segunda fase de veículos são nanopartículas carregadas com drogas ou de agentes de contraste de 6,7. As nanopartículas, que são incorporados no material PSI, estão gradualmente lançado como o Si degrada 8. Uma vantagem de utilizar partículas de Si é que as características de morfologia e de superfície pode ser facilmente ajustada para a obtenção biodistribuição óptimo e libertação da droga. Recentemente, a utilização com sucesso da plataforma MSV para a entrega de lipossomas de siRNA para o tecido do tumor foi demonstrado num modelo de rato do ovário e do cancro da mama 9, 10. Neste trabalho, nós fabricamos um sistema de entrega universal para siRNA com base nos princípios da plataforma MSV. A eficácia deste sistema de entrega tem sido anteriormente demonstrado nósing siRNA diferentes moléculas 11. O sistema é um de silício poroso (PCPS) transportadora funcionalizada-policatião, que consiste em psi enxertado com polietilenimina (PEI) e a arginina (Arg). PEI pode auxiliar na formação de interacções electrostáticas com siRNA, enquanto Arg psi e pode servir para reduzir a toxicidade de PEI, como anteriormente demonstarted 11. Além disso, a presença de PEI pode auxiliar na absorção intracelular e endossomal fuga, ao passo que as micropartículas psi permitir protecção siRNA e de libertação sustentada. As partículas psi degradam gradualmente, sob condições fisiológicas, resultando assim na formação de nanopartículas Arg-PEI / siRNA (Figura 1), que têm uma morfologia distinta e uma distribuição de tamanhos estreita 11. Os dados referentes à estabilidade do sistema PCPS / siRNA, consulte o estudo de Shen et al. 11. Esta plataforma de PCPS difere do MSV convencional, uma vez que as nanopartículas segundo estágio não são inicialmente present no veículo, mas são formados ao longo do tempo como o transportador de primeira fase degrada 11, 12. A eficiência de carregamento de siRNA eficiência, citotoxicidade e silenciamento de genes do sistema PCPS foi avaliada in vitro. A eficiência de transfecção foi medida utilizando siRNA contra a ataxia telangiectasia mutada (ATM) oncogene, que está envolvido na reparação do ADN 10. Previamente, a supressão da ATM foi demonstrado para diminuir o crescimento do tumor em um modelo de cancro da mama 10.

<table border="0" cellpadding="0" cellspacing="0" width="1812">
	<tbody>
		<tr height="55">
			<td height="55" style="height:55px;width:605px;">Name of the Material/Equipment</td>
			<td style="width:421px;">Company/Institution</td>
			<td style="width:133px;">Catalog Number</td>
			<td style="width:653px;">Comments/Description</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Polyethylenimine (PEI), branched</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">408727</td>
			<td style="width:653px;">Average molecular weight ~25,000 Da</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">L-Arginine</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">A5006</td>
			<td style="width:653px;">Reagent grade, &#8805;98%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Boc-Asp-OH</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">408-468</td>
			<td style="width:653px;">99%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">(3-&#8203;Aminopropyl)&#8203;triethoxysilane</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">440140</td>
			<td style="width:653px;">99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Hydrogen peroxide solution</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">216763</td>
			<td style="width:653px;">30&#160;wt. % in H2O</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Sulfuric acid</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">339741</td>
			<td style="width:653px;">100.00%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Isopropyl alcohol</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">W292907</td>
			<td style="width:653px;">&#8805;99.7%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Ethanol</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">459844</td>
			<td>&#8805;99.5%&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride (EDC)</td>
			<td>Sigma-Aldrich</td>
			<td>03449</td>
			<td>&#8805;99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin from bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td>A7030-10G</td>
			<td>Blocking agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Ataxia-telangiectasia mutated siRNA&#160;</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Designed in-house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Tris Acetate-EDTA buffer</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">T9650</td>
			<td style="width:653px;">For DNA agarose gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Penicillin-Streptomycin</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>P4333</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Fetal Bovine Serum</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>F2442</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">TWEEN 20</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>P1379</td>
			<td style="width:653px;">Polyethylene glycol sorbitan monolaurate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">2-Mercaptoethanol</td>
			<td style="width:421px;">Sigma Aldrich</td>
			<td>M6250</td>
			<td style="width:653px;">For Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Sodium dodecyl sulfate</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>L3771</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Sodium phosphate monobasic</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>71496</td>
			<td style="width:653px;">For making phosphate buffer&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Sodium phosphate dibasic</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td>71640</td>
			<td style="width:653px;">For making phosphate buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Mouse IgG</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">A4416</td>
			<td style="width:653px;">Secondary antibody (anti-mouse) for Western blot</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">N-Hydroxysuccinimide (NHS)</td>
			<td style="width:421px;">Sigma-Aldrich</td>
			<td style="width:133px;">130672</td>
			<td style="width:653px;">98%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">CELLSTAR 96W Microplate Tissue Culture Treated Clear w/ Lid</td>
			<td style="width:421px;">Greiner Bio-One</td>
			<td style="width:133px;">655182</td>
			<td style="width:653px;">96-well plate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">10X Tris-Glycine Liquid</td>
			<td style="width:421px;">Li-Cor&#160;</td>
			<td style="width:133px;">928-40010</td>
			<td style="width:653px;">Transfer buffer for Western blot</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Paraformaldehyde solution 4% in PBS&#160;</td>
			<td style="width:421px;">Santa Cruz</td>
			<td>Sc-281692</td>
			<td style="width:653px;">Fixation of cells</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (MTS)</td>
			<td style="width:421px;">Promega</td>
			<td style="width:133px;">G5421</td>
			<td style="width:653px;">Proliferation assay</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Phosphate buffered saline</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td style="width:133px;">BP399-500</td>
			<td style="width:653px;">10 X Solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Corning cellgro Dulbecco&#39;s Modification of Eagle&#39;s (Mod.)</td>
			<td style="width:421px;">Fisher Scientific&#160;</td>
			<td style="width:133px;">MT-15-017-CM</td>
			<td style="width:653px;">Cell culture media, 1X solution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Triton X-100</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td>AC21568-2500</td>
			<td style="width:653px;">Octyl phenol ethoxylate, permeabilization agent</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Cover glass</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td>12-530C&#160;</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Methanol</td>
			<td style="width:421px;">Fisher Scientific&#160;</td>
			<td>A412-1</td>
			<td style="width:653px;">For Western blot transfer buffer</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Plastic Cuvettes</td>
			<td style="width:421px;">Fisher Scientific&#160;</td>
			<td style="width:133px;">14-377-010&#160;</td>
			<td style="width:653px;">For size measurements using Zetasizer Nano ZS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Molecular BioProducts RNase AWAY Surface Decontaminant</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td style="width:133px;">14-754-34</td>
			<td style="width:653px;">Spray for removing RNAse contamination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Agarose</td>
			<td style="width:421px;">Fisher Scientific</td>
			<td style="width:133px;">BP165-25</td>
			<td style="width:653px;">Low melting point, for running RNA samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Gold Antifade Reagent with DAPI&#160;</td>
			<td style="width:421px;">Invitrogen</td>
			<td>P36935</td>
			<td style="width:653px;">Antifade reagent with DAPI, nucelus detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Alexa Fluor 488 Phalloidin</td>
			<td style="width:421px;">Invitrogen</td>
			<td style="width:133px;">A12379</td>
			<td style="width:653px;">Dissolve 300 units in 1.5 ml methanol, detection of filamentous actin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">SYBR Safe DNA Gel Stain</td>
			<td style="width:421px;">Invitrogen</td>
			<td style="width:133px;">S33102</td>
			<td style="width:653px;">Visualization of RNA</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Negative Control siRNA</td>
			<td style="width:421px;">Qiagen</td>
			<td style="width:133px;">1022076</td>
			<td style="width:653px;">Control siRNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">AllStars Neg. siRNA AF 555</td>
			<td style="width:421px;">Qiagen</td>
			<td style="width:133px;">1027294</td>
			<td style="width:653px;">Fluorescent control siRNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Cell scraper</td>
			<td style="width:421px;">Celltreat</td>
			<td>229310</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BioLite Multidishes and Microwell Plates</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td>130184</td>
			<td style="width:653px;">6-well plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce LDS Sample Loading Buffer (4X)&#160;</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td>84788</td>
			<td style="width:653px;">Sample loading buffer for Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Pierce BCS Protein Assay Kit</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td style="width:133px;">23227</td>
			<td style="width:653px;">Protein quantification assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Halt Protease Inhibitor Single-Use Cocktails (100X)</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td style="width:133px;">78430</td>
			<td style="width:653px;">Protease inhibitor cocktail, use at 1X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M-PER Mammalian Protein Extraction Reagent</td>
			<td style="width:421px;">Thermo Scientific&#160;</td>
			<td style="width:133px;">78501</td>
			<td style="width:653px;">Protein extraction reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Sorvall Legend Micro 21R</td>
			<td>Thermo Scientific</td>
			<td>75002440</td>
			<td>Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Beta Actin Antibody&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>MA1-91399</td>
			<td>&#946;-actin primary antibody (from mouse) for western blor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6X TriTrack DNA Loading Dye</td>
			<td style="width:421px;">Thermo Scientific</td>
			<td>R1161</td>
			<td style="width:653px;">DNA loading dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nuclease-Free Water&#160;</td>
			<td style="width:421px;">Life Technologies</td>
			<td>AM9938</td>
			<td style="width:653px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-Fat Dry Milk</td>
			<td style="width:421px;">Lab Scientific</td>
			<td>M0841</td>
			<td style="width:653px;">For Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-well BD Falcon culture slides&#160;</td>
			<td style="width:421px;">BD Biosciences</td>
			<td>354102</td>
			<td style="width:653px;">2-well culture slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amersham ECL Western blot detection reagent.&#160;</td>
			<td style="width:421px;">GE Healthcare Life Sciences</td>
			<td style="width:133px;">RPN2106</td>
			<td style="width:653px;">Western blot detection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BA Membranes</td>
			<td style="width:421px;">GE Healthcare Life Sciences</td>
			<td>10402096</td>
			<td style="width:653px;">Nitrocellulose membrane for Wester blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATM (D2E2) Rabbit mAb</td>
			<td style="width:421px;">Cell Signaling</td>
			<td>2873S</td>
			<td style="width:653px;">ATM primary antibody (from rabbit) for Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-rabbit IgG, HRP-linked Antibody</td>
			<td style="width:421px;">Cell Signaling&#160;</td>
			<td>7074</td>
			<td style="width:653px;">Secondary antibody (anti-rabbit) for Western blot&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Folded capillary cells</td>
			<td style="width:421px;">Malvern&#160;</td>
			<td style="width:133px;">DTS 1061</td>
			<td style="width:653px;">For zeta potentail measurements using Zetasizer Nano ZS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">MDA-MB-231 cell line</td>
			<td style="width:421px;">ATCC</td>
			<td style="width:133px;">HTB-26</td>
			<td style="width:653px;">Mammary Gland/Breast</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">12% Mini-PROTEAN TGX Gel</td>
			<td>Bio-rad</td>
			<td>456-1043&#160;</td>
			<td style="width:653px;">For Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biorad PowerPac HC</td>
			<td>Bio-rad</td>
			<td>164-5052</td>
			<td>Power supply for electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x Tris/Glycine/SDS Buffer</td>
			<td>Bio-rad</td>
			<td>161-0732</td>
			<td>Running buffer for Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wide Mini-Sub Cell GT Cell</td>
			<td>Bio-rad</td>
			<td>170-4405</td>
			<td>Electrophoresis equipment for DNA agarose gel&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-PROTEAN Tetra cell</td>
			<td>Bio-rad</td>
			<td>165-8000</td>
			<td>Electrophoresis equipment for Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChemiDoc XRS+ System with Image Lab Software</td>
			<td>Bio-rad</td>
			<td>170-8265</td>
			<td>Image acquisition and analysis software for gels and blots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#34; (10cm) dia., 5x7mm diced Silicon Wafer</td>
			<td>Ted Pella&#160;</td>
			<td>16007</td>
			<td>Silicon waferfor scanning electron microscopy and atomic force microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermomixer R</td>
			<td>Eppendorf</td>
			<td>22670107</td>
			<td>Shaker&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoton II diluent</td>
			<td>Beckman Coulter</td>
			<td>8546719</td>
			<td>Isoton diluent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multisizer 4 Coulter Counter</td>
			<td>Beckman Coulter</td>
			<td>A63076</td>
			<td>Particle counting analyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Non-functionalized porous silicon particles&#160;</td>
			<td style="width:421px;">Microelectronics Research Center, University of Texas at Austin</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Dicoidal shape. 2.6 &#956;m (diameter) x 0.7 &#956;m (hight), provided in isopropyl alcohol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Zetasizer Nano ZS</td>
			<td style="width:421px;">Malvern</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Particle analyzer system for size and zeta potential&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Scanning Electron Microscope</td>
			<td style="width:421px;">FEI</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Particle size and shape&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:605px;">Atomic Force Microscope</td>
			<td style="width:421px;">Bruker</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Particle size and shape</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:605px;">Fluo ViewTM 1000 Confocal Microscope</td>
			<td style="width:421px;">Olympus</td>
			<td style="width:133px;"></td>
			<td style="width:653px;">Visualization of fixed and live cells</td>
		</tr>
	</tbody>
</table>

porous silicon microparticles for delivery of sirna therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Este protocolo descreve o uso de um sistema de entrega n viral para a segura e eficiente transfecção siRNA. Os resultados de SEM revelam que as partículas de PCPS são de forma cilíndrica e têm um diâmetro de 2,6 mm (Figura 2A). As partículas são carregadas positivamente com um potencial zeta de cerca de 8,21 (Figura 2B), permitindo desse modo electrostático de ligação com os nucleótidos carregados negativamente. Imagens confocais de diferentes camadas de partículas de PCPS...

Watch this Scientific Journal Video about Porous Silicon Microparticles for Delivery of siRNA Therapeutics at JoVE.com

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Entrega continua a ser o principal desafio para a aplicação terapêutica de RNA de interferência pequeno (siRNA). Este protocolo envolve a utilização de uma plataforma de entrega de siRNA multifuncional e biocompativel, que consiste de arginina e polietilenoimina enxertado micropartículas de silício poroso.

Porous Silicon micropartículas para entrega de siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

porous-silicon-microparticles-for-delivery-of-sirna-therapeutics

Research

JoVE Journal

Bioengineering

10.8K visualizações.  Houston Methodist Research Institute.  Entrega continua a ser o principal desafio para a aplicação terapêutica de RNA de interferência pequeno (siRNA). Este protocolo envolve a utilização de uma plataforma de entrega de siRNA multifuncional e biocompativel, que consiste de arginina e polietilenoimina enxertado micropartículas de silício poroso. 

Vídeo: Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

Funcionalização litografia macia e livre de óxido Patterning silício e germânio

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

Bioengenharia

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Andaimes auto-relato para cultura de células 3-Dimensional

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we describe the preparation of polycrystalline silicon nanowire field-effect transistors (pSNWFETs) that serve as biosensing devices for biomarker detection. A protocol for chemical and biomolecular sensing by using pSNWFETs is presented. The pSNWFET device was demonstrated to be a promising transducer for real-time, label-free, and ultra-high-sensitivity biosensing applications. The source/drain channel conductivity of a pSNWFET is sensitive to changes in the environment around its silicon nanowire (SNW) surface. Thus, by immobilizing probes on the SNW surface, the pSNWFET can be used to detect various biotargets ranging from small molecules (dopamine) to macromolecules (DNA and proteins). Immobilizing a bioprobe on the SNW surface, which is a multistep procedure, is vital for determining the specificity of the biosensor. It is essential that every step of the immobilization procedure is correctly performed. We verified surface modifications by directly observing the shift in the electric properties of the pSNWFET following each modification step. Additionally, X-ray photoelectron spectroscopy was used to examine the surface composition following each modification. Finally, we demonstrated DNA sensing on the pSNWFET. This protocol provides step-by-step procedures for verifying bioprobe immobilization and subsequent DNA biosensing application.

We describe key steps for biosensing by using polysilicon nanowire field-effect transistors, including the preparation of the device and the immobilization and confirmation of a DNA molecular probe on the nanowire surface, as well as conditions for DNA sensing.

Preparação de Silicon Nanowire transistor de efeito de campo para aplicações químicas e Biossensoriais

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

Síntese de termogelificantes poli (N-isopropilacrilamida) -graft-condroitina Composites Sulfato com alginato micropartículas para Engenharia de Tecidos

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

Entrega de terapêutica siRNA para o SNC Usando catiônicos e aniônicos lipossomas

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

Projetar filmes de silício poroso como portadores do fator de crescimento do nervo

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

3D impresso celulose porosa nanocompósitos Hydrogel andaimes

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

Preparação de nanopartículas poliméricos neutralmente carregadas e responsivas ao pH para entrega de siRNA citosólica

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

Nanofios de Silício e Estimulação Óptica para Investigações de Acoplamento Elétrico Intra e Intercelular

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These ...

Tools for Surface Treatment of Silicon Planar Intracortical Microelectrodes

Intracortical microelectrodes hold great therapeutic potential. But they are challenged with significant performance reduction after modest implantation durations. A substantial contributor to the observed decline is the damage to the neural tissue proximal to the implant and subsequent neuroinflammatory response. Efforts to improve device longevity include chemical modifications or coating applications to the device surface to improve the tissue response. Development of such surface treatments is typically completed using non-functional "dummy" probes that lack the electrical components required for the intended application. Translation to functional devices requires additional consideration given the fragility of intracortical microelectrode arrays. Handling tools greatly facilitate surface treatments to assembled devices, particularly for modifications that require long procedural times. The handling tools described here are used for surface treatments applied via gas-phase deposition and aqueous solution exposure. Characterization of the coating is performed using ellipsometry and x-ray photoelectron spectroscopy. A comparison of electrical impedance spectroscopy recordings before and after the coating procedure on functional devices confirmed device integrity following modification. The described tools can be readily adapted for alternative electrode devices and treatment methods that maintain chemical compatibility.

The present protocol describes tools for handling silicon planar intracortical microelectrodes during treatments for surface modification via gas deposition and aqueous solution reactions. The assembly of the components used to handle the devices throughout the procedure is explained in detail.

Ferramentas para tratamento superficial de microeletroletos intracorticais planar de silício

Intracortical microelectrodes hold great therapeutic potential. But they are challenged with significant performance reduction after modest implantation ...