Research reported in this publication was supported by The Johns Hopkins Applied Physics Laboratory&#8217;s Research and Exploratory Development Department, Office of Technology Transfer, and Stuart S. Janney Fellowship Program, in addition to the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number R21HL127355.

1. Processamento de SLNs <ol><li> Síntese de SLNs Nota: Use o método PIT 1,17,20 para preparar os sub-20 SLNs NM 1. <ol><li> Utilizar técnicas assépticas, biorreagentes ou reagentes de grau de cultura de células, e materiais estéreis (isto é, pipetas, espátulas, ampolas, etc.). </li><li> Usar uma cabine de segurança biológica para a síntese de NLS (Figura 2). </li><li> Combinar 0,6 mg de corante fluorescente (Vermelho do Nilo (NIR), a concentração de NiR é de aproximadamente 0,3 mg / ml) e 0,10 g de heneicosano (lípido, 5% p / p de concentração de lípido) para um frasco (15 ml), o co-fundido a 90 ° C, e mexa. </li><li> Adicionar 0,11 g de polioxietileno (10) éter oleilico (surfactante, 5,5% p / p de concentração de surfactante). A co-fundir a mistura resultante a 90 ° C e agitar. </li><li> Adicionar 1,79 g de água estéril à mistura, aquecer a 90 ° C, e agita-se até que uma nanoemulsão transparente é formado. Nota: Sob agitação contínua e arrefecimento controlado, SLNs são criados. Estas condições causar a inversão da emulsão de água-em-óleo para uma emulsão óleo-em-água. Moléculas lipofílicas como partição NiR para as gotículas lipídicas. </li><li> Prepara-se uma nanoemulsão em paralelo sem a adição do corante fluorescente, a fim de servir como amostra de controlo. Combinar 0,10 g de heneicosano e 0,11 g de polioxietileno (10) éter oleílico para um frasco (15 ml) co-fundido a 90 ° C, e agita-se. <ol><li> Adicionar 1,79 g de água estéril à mistura, aquecer a 90 ° C, e agita-se até que uma nanoemulsão transparente é formado. </li></ol></li><li> Além disso esterilizar cada nanoemulsão utilizando um filtro estéril de 0,2 um. </li></ol><img alt="Figura 2" src="/files/ftp_upload/53102/53102fig2.jpg" /> Figura 2. Esquema de síntese de SLNs. <a href="https://www.jove.com/files/ftp_upload/53102/53102fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> </li><li> Tamanho da Partícula e Polydispersity <ol><li> Use dispersão de luz dinâmica (DLS) para medir o tamanho de partícula e polidispersidade dos NLS 1 utilizando uma cuvete de vidro e um instrumento concebido para medir o tamanho de partícula. </li><li> Antes das medições de amostras, medir as dimensões das partículas de dois padrões conhecidos seguindo o protocolo do fabricante para a preparação de padrões e de medição. </li><li> Encher a cuvete de vidro com 1,5 ml de solução padrão e a medição da solução padrão, utilizando as mesmas condições de medida que as amostras. Nota: passo anterior foi repetido para cada padrão. </li><li> Medem-se as amostras tal como preparado. Para cada, utilizar os seguintes parâmetros experimentais: um tempo de funcionamento de 100 seg, índice de refracção de água (1,33), a viscosidade da água a 20 ° C (1,002 mPa). Nota: Esta técnica utiliza uma frequência deslocada luz para medir o tamanho das nanopartículas. </li><li> Relate o diâmetro médio das partículas e polidispersividade de partidistribuição de tamanho de cle. </li></ol></li><li> Ponto de fusão e Calor Latente de fusão <ol><li> Use um calorimetria exploratória diferencial (DSC) equipado com um amostrador automático e fonte de resfriamento de nitrogênio líquido para investigar o comportamento térmico de SLNs 1. </li><li> Pipetar os SLNs como preparados em uma panela de alumínio de 40 l com uma massa de cerca de 25 mg e fechar hermeticamente a panela utilizando uma prensa crimper universal para minimizar a perda de umidade durante a varredura DSC. </li><li> Medir cada nanoemulsão de 5 a 80 ° C. </li><li> Indica-se a ponto de fusão e do calor latente de fusão de DSC a partir do lote, em que o vale representa o ponto de fusão e o integral da área sob a curva de DSC na trama dividida pela quantidade de material representa o calor latente de fusão. </li></ol></li></ol> 2. SLNs Interação com fibroblastos e células dendríticas <ol><li> Cultura de fibroblastos humanos primários <ol><li> Cultura primária fi humanafibroblastos de acordo com as instruções do fabricante em meio líquido para a cultura de fibroblastos dérmicos humanos (média 106), suplementado com o kit de suplemento de crescimento de soro baixo (LSGS). Manter as células numa atmosfera humidificada a 37 ° C, 5% de CO 2 e subcultura, ao atingir 80% de confluência. </li><li> Para tanto MTT e análise de imagem, as células de semente em uma placa de 96 poços, a uma densidade de 2 x 10 4 culas por po, e deixar equilibrar a 37 ° CO / N antes da exposição SLN. </li><li> No tempo zero, diluir SLNs em meio completo, conforme indicado (Figura 4), ​​com respeito à concentração de lípido, e adicionar 10 uL por poço para cada replicado poço na placa de 96 poços. </li><li> Manter as células a 37 ° C, 5% de CO 2, durante o período de incubação. </li></ol></li><li> MTT Toxicidade Assay <ol><li> Após 24 h de incubação com o NLS, determinar a viabilidade celular utilizando o ensaio de MTT, de acordo com o protocolo do fabricante. &lt;/ li&gt; <li> Lave as células uma vez e adicionar meio fresco a cada poço (100 ul / poço). Matar poços de controlo por incubação numa solução de metanol a 70% durante 10 min antes de substituir com meio fresco. </li><li> Adiciona-se reagente de MTT (10 ul por poço) a cada poço da microplaca e incube O / N a 37 ° C. </li><li> Depois de O / N de incubação, os cristais de formazina solubilizar intracelulares com a solução detergente fornecida (100 ul por poço) de acordo com o protocolo do fabricante. </li><li> Após 3 h de incubação à TA, obter valores de absorvância a um comprimento de onda de medição de 570 nm utilizando um leitor de microplacas. O MTT utilizado deve ser menor do que 1% (w / v) de 3- (4,5-dimetiltiazolil-2) -2, brometo de 5-difenil-tetrazólio: Nota. </li></ol></li><li> Microscopia de Fluorescência <ol><li> Em pontos de tempo após a dosagem específicos de fibroblastos com SLN, lavar os fibroblastos duas vezes com solução salina tamponada com fosfato estéril (PBS) e fixar durante 10 min com metanol a 70% frio. </li><li> Após 10 min, Substituir o metanol com PBS e capturar imagens usando um microscópio invertido, usando uma passagem de banda (PA) 690/50 nm conjunto de filtros. </li></ol></li><li> Cultura de Medula Óssea de rato As células dendríticas <ol><li> Induzem as células dendríticas derivadas de medula óssea de ratinho (BMDC) como previamente descrito 23,24. Resumidamente, suspensões de placa única de células de ratinhos C57BL / 10 de medula óssea em 100 mm de placas de Petri a 2 x 10 6 / placa com factor estimulador de colónias de granulócitos-macrófagos de murino recombinante (GM-CSF, 300 U / ml) e murino IL-4 recombinante (factor de células B, 200 U / ml de estimulante). Mudança de mídia a cada 3 dias. </li><li> No dia 7, adicionar SLNs NiR-carregadas com os pratos a uma concentração final de 0,5 e 5,0 ug / ml de lípido e manter as células em uma temperatura de 37 ° C, 5% de CO2 para o comprimento desejado de exposição. </li><li> Coletar as células dos pratos por aspiração de toda a mídia na placa, cobra-lo em um tubo de 50 ml, seguido de lavagem completa da placa (para desalojar anúncio vagamenteHerent células) com 5 ml de solução salina tamponada com fosfato (PBS) contendo 4 mM de ácido etilenodiaminotetracético (EDTA). Recolha a suspensão celular obtida no mesmo tubo de 50 ml. </li></ol></li><li> Avaliação da SLNs Incorporação via Citometria de Fluxo <ol><li> Determinar a concentração da suspensão de células obtida no ponto 2.4.3 e distribuir 3x10 5 células em um de 12 mm x 75 mm de fundo redondo tubo FACS. </li><li> Lavam-se as células por adição de 1 ml de PBS e em seguida os tubos de centrifugação durante 5 minutos a 400 xg, à temperatura de 4 ° C. </li><li> Descartar o sobrenadante e ressuspender o sedimento com 100 ul de PBS / EDTA 2 mM / 0,1% de albumina de soro bovino (BSA) (tampão de coloração) contendo anticorpo monoclonal anti-CD11c-FITC (0,25 ug / mL de concentração final). Incubar durante 30 min em gelo no escuro. </li><li> Lavam-se as células com 1 ml de PBS e, em seguida, centrifugar os tubos durante 5 min a 400 xg, à temperatura de 4 ° C. </li><li> Descartar o sobrenadante e ressuspender cadasedimentar com 400 ul de tampão de coloração. As células estão agora prontos para a aquisição das amostras com um citômetro de fluxo (possivelmente equipado com lasers azuis e vermelhas). </li><li> Utilizando um software de análise de citometria de fluxo, avaliar o nível de incorporação de NLS por DC por gating na população de células FITC CD11c + (positivo) e comparando a intensidade de fluorescência NLS-associados (medido nos canais PerCP-Cy5.5 ou APC). </li></ol></li></ol>

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The authors have nothing to disclose.

Neste estudo, a síntese de NLS e a sua aplicabilidade para aplicações destinadas intracelulares foram exploradas. Estas nanopartículas biocompatíveis têm mostrado promissores como veículos de entrega para várias aplicações, incluindo a entrega de drogas, o silenciamento do gene, e tecnologias de vacina 25-30. Ultra-pequenas SLNs foram sintetizados usando um processo fácil, e as suas interacções com células epiteliais primárias e células imunitárias primárias foram exploradas. SLNs foram conc...

Veículos de entrega baseado em nanopartículas têm mostrado uma grande promessa para aplicações dirigidas intracelulares, fornecendo um mecanismo para alterar especificamente sinalização celular e expressão gênica. Estes veículos podem ser carregados com medicamentos, proteínas, ácidos nucleicos e concebidos para influenciar as respostas celulares e conseguir um efeito desejado em tecidos-alvo. Muitos tipos de nanocarriers têm sido exploradas para benefício terapêutico e de diagnóstico, incluindo lipidos, polímeros, silício e materiais magnéticos. Estes sistemas são atraentes devido ao seu potencial para a entrega da droga localizada, aumento da concentração terapêutica em tecidos-alvo e redução de toxicidade sistêmica. Nanopartículas de lípidos sólidas (NLS) são um exemplo bem estudado de um sistema de entrega de nanopartículas que surgiu como um veículo de entrega de droga promissora nos últimos anos. SLNs pode ser facilmente formulado para várias aplicações, incluindo bio-sensor 1, cosméticos 2, e tentrega herapeutic 3-7. A sua utilidade advém do facto de que eles são constituídos inteiramente de reabsorvíveis, lípidos não tóxicos, resultando em melhor biocompatibilidade. Durante a síntese, os fármacos lipofilicos podem ser incorporados em veículos SLN, aumentando assim a solubilidade do fármaco e da aptidão para administração parentérica. SLN veículos também ajudam a estabilizar terapêuticas encapsuladas, reduzindo sua degradação e depuração, e maximizando a ação terapêutica. Estes veículos são particularmente bem adequados para longa actuação, preparações de libertação controlada devido à sua estabilidade à temperatura do corpo 3,4,8,9. Importante, a encapsulação de fármacos em nanopartículas de lípidos altera os perfis farmacocinéticos intrínsecas das moléculas de fármaco. Isto proporciona uma vantagem potencial de permitir a libertação controlada de fármacos com um índice terapêutico estreito. A taxa de libertação de agentes terapêuticos incorporados-NLS pode ser regulado com base na taxa de degradação do lípido ou a taxa de difusão da droga namatriz lipídica. SLNs são muitas vezes modificados para se acumular nos tecidos alvo específicos. Por exemplo, o tamanho (tipicamente maior do que 10 nm) potencia a retenção na circulação, em que a vasculatura de tecido tumoral gotejante facilita a deposição. Além disso, a via de administração da partícula foi mostrado para alterar a biodistribuição com o potencial para segmentar estruturas fisiológicas específicas tais como os nódulos linfáticos 10,11. Após a deposição nos tecidos alvo, conseguindo interacções celulares apropriados e eventual internalização de nanopartículas é um desafio devido à capacidade das membranas celulares para controlar selectivamente o fluxo de iões e moléculas para dentro e para fora da célula 12. Para facilitar a absorção celular, é possível modificar nanocarriers com ligandos específicos, incluindo péptidos, moléculas pequenas, anticorpos monoclonais e 13,14. Vários mecanismos incluindo tanto passiva penetração e transporte activo de nanopartículasatravés da membrana celular foram previamente descritos 3,12,15. Em geral, demonstrou-se que as interacções célula-nanopartículas são influenciadas pelas propriedades físico-químicas das nanopartículas, incluindo o tamanho, forma, carga de superfície e a química de superfície, além de parâmetros específicos de células, tais como tipo de célula ou a fase do ciclo celular 12. Uma investigação anterior demonstrou a síntese de sub-10 SLNs nm para 16 tópicos e aplicações de detecção de biomarcadores 1 utilizando o método de 17 temperatura de inversão de fase (PIT). Este é um método de síntese 2 suave onde a composição permanece constante enquanto a temperatura é gradualmente alterado. Agitação contínua da solução aquecida, à medida que arrefece a resultados de RT em uma nanoemulsão. Este processo resulta na síntese de NLS com tamanho de partícula menor do que 1 relatado anteriormente usando vários métodos para a síntese do lípido nanoparticles 17-22. A escala de tamanho resultante, menos de 20 nm, proporciona uma vantagem para aplicações de segmentação intracelulares devido ao aumento da área superficial eo potencial para interações celulares avançados. Um esquema de NLS, concebido para proporcionar um corante fluorescente ou terapêutico, é mostrado na Figura 1. Os SLNs consistem de um lípido interior (por exemplo, alcano linear), que permite a incorporação de compostos lipófilos (por exemplo, corantes ou agentes terapêuticos) e um exterior surfactante (surfactante não iónico por exemplo, linear) rodeada por água. Neste estudo, SLNs foram carregadas com um corante fluorescente e utilizado como um modelo para investigar interacções célula-partículas. Fibroblastos dérmicos humanos primários e células dendríticas de rato foram expostos a corante carregado-NLS ao longo do tempo, a fim de caracterizar as interacções com respeito à toxicidade e a absorção das partículas. Um grupo 3- (4,5-dimetiltiazol-2-il) -2,5-diphenylphenyltetrazolium (MTT) foi utilized, a fim de estabelecer níveis de dosagem adequados. A microscopia de fluorescência e citometria de fluxo foram utilizados dois métodos para analisar a absorção de partículas in vitro. <img alt="figura 1" src="/files/ftp_upload/53102/53102fig1.jpg" /> Figura 1. Esquema da SLN mostrando os principais constituintes. <a href="https://www.jove.com/files/ftp_upload/53102/53102fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Nile Red (NiR)</td>
			<td>Sigma</td>
			<td>19123</td>
			<td>BioReagent, suitable for fluorescence, &#8805;98.0%</td>
		</tr>
		<tr>
			<td>Heneicosane</td>
			<td>Aldrich</td>
			<td>286052</td>
			<td>98%</td>
		</tr>
		<tr>
			<td>Brij O10</td>
			<td>Sigma</td>
			<td>P6136</td>
			<td>Brij 97, C18-1E10, Polyoxyethylene (10) oleyl ether</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W3500</td>
			<td>Sterile-filtered, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Syringe Filter 0.2 &#181;m Supor Membrane Low Protein Binding</td>
			<td>Life Sciences</td>
			<td>PN4612</td>
			<td>Non-Pyrogenic</td>
		</tr>
		<tr>
			<td>Nanotrac Ultra&#160;</td>
			<td>Microtrac</td>
			<td>serial number U1985IS</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>Differential Scanning Calorimeter</td>
			<td>Mettlet-Toledo</td>
			<td>----</td>
			<td>Instument</td>
		</tr>
		<tr>
			<td>Primary human fibroblasts&#160;</td>
			<td>Life Technologies</td>
			<td>C-004-5C</td>
			<td>Neonatal (HDFn)</td>
		</tr>
		<tr>
			<td>Medium 106</td>
			<td>Life Technologies</td>
			<td>M-106-500</td>
			<td>A sterile, liquid medium for the culture of human dermal fibroblasts.</td>
		</tr>
		<tr>
			<td>Low Serum Growth Supplement Kit (LSGS Kit)</td>
			<td>Life Technologies</td>
			<td>S-003-K</td>
			<td>All the components of complete LSGS</td>
		</tr>
		<tr>
			<td>MTT Cell Proliferation Assay Kit</td>
			<td>Trevigen</td>
			<td>4890-025-K</td>
			<td>Sensitive kit for the measurement of cell proliferation based upon the reduction of the tetrazolium salt, 3,[4,5-dimethylthiazol-2- yl]-2,5-diphenyl-tetrazolium bromide (MTT)</td>
		</tr>
		<tr>
			<td>Safire2 microplate reader</td>
			<td>Tecan</td>
			<td>----</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline&#160;</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td>For molecular biology</td>
		</tr>
		<tr>
			<td>Recombinant murine GM-CSF&#160;</td>
			<td>R&#38;D Systems</td>
			<td>415</td>
			<td>&#62;97%, by SDS-PAGE under reducing conditions and visualized by silver stain.</td>
		</tr>
		<tr>
			<td>Recombinant murine IL-4&#160;</td>
			<td>R&#38;D Systems</td>
			<td>404</td>
			<td>&#62;97%, by SDS-PAGE under reducing conditions and visualized by silver stain.</td>
		</tr>
	</tbody>
</table>

solid lipid nanoparticles (slns) for intracellular targeting applications

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

O método PIT foi utilizado para sintetizar os NLS e a temperatura de inversão de fase foi determinada utilizando um banho de água. As amostras foram aquecidas lentamente e agitado suavemente até que a solução apareceu clara. A temperatura de inversão de fase para as SLNs feitas usando heneicosano lipídica é de 45 ° C. A Tabela 1 sumariza o tamanho de partícula, polidispersão, ponto de fusão e do calor latente de fusão para os NLS. Os NLS sintetizados utilizando as condições de processame...

Watch this Scientific Journal Video about Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications at JoVE.com

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

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

Nanopartículas Lipídicas Sólidas (NLS) para intracelular Segmentação Aplicações

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

solid-lipid-nanoparticles-slns-for-intracellular-targeting

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JoVE Journal

Bioengineering

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

16.8K visualizações.  The Johns Hopkins Applied Physics Laboratory. In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions. 

Vídeo: Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

Nanopartículas virais para<em&gt; Em vivo</em&gt; Imaging Tumor

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Bioengenharia

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Espremendo celular como uma plataforma de entrega intracelular Robust Microfluidic

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

Harmonic Nanopartículas Regenerativa Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

Produção e Segmentação de Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Bioativa de Prussian Blue Nanopartículas para Multimodal Imagem Molecular Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

Rotulagem celular e Segmentação com nanopartículas de óxido de ferro superparamagnéticas

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

Fabricação e entrega da droga Aplicações de seda Nanopartículas

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Captação de novas nanopartículas revestidos de lipídeos contendo Falcarindiol por células-tronco mesenquimais humanas

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

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 ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

Formulando e caracterizando nanopartículas lipídicas para entrega de genes usando uma plataforma de mistura microfluida

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...