This work was supported by the following grants: NIH-SC3 grant, NSF-PREM grant, Hampton University Faculty Research Grant. We would like to thank Mrs. Michele A. Cochran at Virginia Institute of Marine Science (VIMS) for the use of the particle size analyzer. We would also like to thank Mrs. Corinne R. Ramaley for reviewing the manuscript.

1. Síntese de DOX-PA <ol><li> Pesar 390 mg de doxorrubicina e 243 mg de hidrazida de ácido palmítico, e transferir para um balão de fundo redondo. </li><li> Adicionar 150 ml de metanol anidro para o balão com uma seringa de vidro. Adicionar 39 ul de ácido trifluoroacético (TFA), com uma pipeta. Usando um agitador magnético, agita-se a mistura de reacção durante 18 horas à temperatura ambiente no escuro. NOTA: As quantidades de materiais de reacção podem ser ampliados ou reduzidos para se obter diferentes quantidades de DOX-PA. A proporção dos reagentes deve ser mantida nas mesmas proporções. As reacções que utilizam quantidades de DOX na gama de 78 mg a 1170 mg pode ser realizado num laboratório normal química. </li><li> Purificação de DOX-PA usando uma coluna de gel de sílica. 17 <ol><li> Remover o solvente na mistura de reacção com um evaporador rotativo. Adicionar 3 g de gel de sílica após o volume da mistura é reduzida para cerca de 20 ml. Continue a evaporação rotativa para se obter pós secos e para permitir que tele adsorção de produtos sobre o gel de sílica. Manter a amostra sob vácuo durante mais 30 min após os pós secos são formados. </li><li> Pacote de 50 g de gel de sílica numa coluna usando diclorometano como solvente. Cuidadosamente adicionar a amostra de gel de sílica contendo o produto adsorvida para a coluna. </li><li> Elui-se a coluna com uma mistura de diclorometano e metanol, aumentando gradualmente a percentagem de metanol, aumentando assim a polaridade do solvente (Tabela 1). </li><li> Recolha fracções de eluente em tubos de ensaio (25 ml / tubo) e monitorizar o progresso por cromatografia de camada fina (TLC). </li><li> Combinar todas as fracções contendo DOX-PA puro e remover o solvente usando um evaporador rotativo até o pó seca é formada. Além disso secar o produto sob um vácuo O / N. </li></ol></li><li> Análise de DOX-PA por TLC. <ol><li> Corte um x 8 cm secção de 4 cm de placa de TLC. soluções de amostra spot 0,5 cm da parte inferior da placa com TLC capilares mancha usando conheceuhanol como o solvente. </li><li> Colocar a placa de TLC para uma câmara de desenvolvimento contendo uma mistura de diclorometano e metanol (3/1, v / v). A profundidade do solvente deve ser apenas menos de 0,5 cm. </li><li> Remova a placa da câmara de revelação quando a frente de solvente atingir o topo da placa. Marcar a localização da frente de solvente com um lápis e permitem que a placa secar. Colocar a placa de TLC para uma câmara de coloração contendo vapor de iodo saturada, a fim de visualizar amostras. </li></ol></li><li> Análise de DOX-PA com um H-Nuclear Magnetic Resonance Spectroscopy (1H-RMN). 18 <ol><li> Dissolve-se 15 mg de DOX-PA em 1 ml de sulfóxido de metil-d6 (DMSO) e transferir a amostra para um tubo de RMN. </li><li> Insira o tubo de RMN para o ímã do instrumento de RMN. Medir o espectro de protões, seleccionando de DMSO como um solvente. Retire o tubo de RMN do ímã. Analise o resultado RMN 18. </li></ol></li></ol> 2. Preparação de micelas de DOX-PA pelo método de película-dispersão <ol><li> Dissolve-se DSPE-PEG (40 mg) e DOX-PA (4 mg) com 2 ml de metanol em um frasco de vidro de 10 ml. </li><li> Remover o solvente orgânico sob vácuo utilizando um evaporador rotativo, até se formar um filme fino no frasco. NOTA: Como alternativa, evapora-se os solventes orgânicos sob gás inerte (por exemplo, árgon ou azoto gasoso) para formar uma película e manter o frasco em um exsicador de vácuo para remover adicionalmente o solvente residual. </li><li> Transferência de 2 ml de solução salina de Dulbecco tamponada com fosfato (pH 7,4, DPBS) para o frasco de vidro. </li><li> Colocar o frasco num banho de ultra-sons durante 3 minutos à temperatura ambiente para gerar micelas. NOTA: A energia de ultra-som varia entre os diferentes modelos de banhos ultra-sônicos. Seleccionar uma unidade que pode gerar energia ultra-sónica suficiente para dispersar o filme de polímero / fármaco fina. O poder de banho ultra-sônico usado neste protocolo de saída é de 110 W. </li><li> Manter micelas a 4 ° C para armazenagem de curta duração e -20 ° C durante longoarmazenamento -termo. NOTA: Como alternativa, micelas também podem ser liofilizados e reconstituídos com água antes da utilização. Normalmente, não cryoprotectant ou lioprotector é necessário para esta formulação. </li></ol> 3. Caracterização de DOX-PA As micelas <ol><li> Determinação da concentração de DOX-PA em micelas e eficiência de encapsulação de drogas <ol><li> Dissolve DOX-PA sintetizados nos passos anteriores em DMSO para se preparar soluções de DOX-PA de cinco concentrações diferentes: 1 ug / ml, 5 ug / ml, 20 ug / ml, 50 ug / ml, e 100 ug / ml. Medir a absorção de soluções de DOX-PA com um espectrómetro de UV-VIS a 490 nm. Gerar uma curva padrão baseada nas concentrações de droga DOX-PA e o seu correspondente de absorção a 490 nm. </li><li> Diluir 25 uL de micelas carregadas com fármaco com 500 ul de DMSO. Medir a absorção a 490 nm com um espectrómetro UV-VIS. Calcule as concentrações de droga com a curva padrão gerada no ponto 3.1.1. </li><li> Calcular a eficiência de encapsulação usando a seguinte equação: Eficiência de encapsulamento de droga (%) = (quantidade de drogas em micelas) / (quantidade de fármaco adicionado) x 100% </li></ol></li><li> Caracterização do tamanho de partícula com dispersão de luz dinâmica (DLS) <ol><li> Diluir micelas com DPBS (pH 7,4) até uma concentração final de DSPE-PEG de 1 mg / ml. Analisar uma amostra de 2 ml com um analisador de tamanho de partículas para se obter o tamanho e a polidispersidade índice médio Z (PDI). </li></ol></li><li> Avaliação in vitro actividade anti-cancro NOTA: Use técnica estéril apropriada e operar dentro de uma cabine de segurança biológica. <ol><li> Remover o meio de cultura celular a partir de um frasco de cultura de células (por exemplo, T25) contendo células DU-145 de cancro da próstata humano e lavar as células com 2 mL de DPBS (pH 7,4). </li><li> Aspirar DPBS, adicionar 1 ml de solução de tripsina (0,25%), e incuba-se durante 2 min a 37 ° C para separar as células. </li><li> Adicionar 10 ml de meio de cultura celular (RPMI 1640 + 10% de Soro Fetal Bovino + 1% de antibiótico-antimicótico), quando a maioria das células são separadas do balão. Transferir as células para um tubo de centrífuga de 15 ml e centrifugar as células a 1000 xg durante 5 min. </li><li> Re-suspender o sedimento de células com 5 ml de meio de cultura celular e remover uma amostra para a contagem do número de células com um hemocitómetro. Dilui-se as células com meio de cultura celular a uma densidade de 50.000 células / ml. Adicionar as suspensões de células diluídas em uma placa de cultura celular de 96 poços (100 ul / poço). Incubam-se as células numa incubadora de cultura de células (37 ° C, 5% CO 2) durante 18 h, para permitir a fixação das células. </li><li> Diluir DOX dimetil sulfóxido solução (DMSO) e solução de DOX-PA DMSO com meio de cultura de células para se obter concentrações finais da droga de 0,1 uM, 0,5 uM, 2 uM, 5 uM, 10 uM e, respectivamente. Manter a concentração final de DMSO em todas as amostras acima de 0,5%. Diluir micela DOX-PA com meio de cultura de células para se obter co farmacológico finalncentrations de 0,1 uM, 0,5 uM, 2 uM, 5 uM, 10 uM e, respectivamente. Utilizar o meio de cultura de células em branco um controlo. </li><li> Remova a placa de cultura celular de 96 poços a partir do incubador e substituir o meio de cultura de células com 100 ul de meio contendo diferentes agentes de tratamento preparado no passo 3.3.5 (n = 4 para cada grupo). Incubam-se as células na incubadora de cultura de células (37 ° C, 5% CO 2) durante uma 72 horas adicionais. </li><li> Aspirar o meio e adicionar 100 uL de meio contendo 0,5 mg / ml de 3- (4,5-dimetil-tiazol-2-il) -2,5-difenil (MTT). </li><li> Incubam-se as células na incubadora de cultura de células durante mais 2 h. Remova cuidadosamente o meio e adicionar 100 ul de DMSO para dissolver cristais de formazano. </li><li> Medir a absorvância com o espectrofotómetro de microplacas a um comprimento de onda de 570 nm e um comprimento de onda de referência de 670 nm. </li><li> Calcula-se a viabilidade celular utilizando a seguinte equação: <strong&gt; Viabilidade celular (%) = (A Teste / controlo) × 100% NOTA: Comparar a viabilidade celular entre os diferentes grupos usando uma análise de uma via de variância (ANOVA) teste estatístico. Calcula-se a CI 50 com base na viabilidade celular comparada dados de concentração de droga. </li></ol></li></ol>

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The authors have nothing to disclose.

Neste trabalho, nós descrevemos, um método de filme rápida dispersão simples para a preparação de micelas. Este método utiliza as propriedades de auto-montagem de um polímero anfifílico (por exemplo, DSPE-PEG) para formar micelas com núcleo e camadas estruturadas num ambiente aquoso. Este método de preparação de micelas tem várias vantagens. 1. Trata-se de um processo de formulação simples, o que evita a utilização de passos de redução de tamanho (complicados, tais como extrusão ou homogene...

A quimioterapia é utilizada para tratar várias formas de cancro. A maioria, se não todos, os fármacos de quimioterapia têm efeitos secundários tóxicos que podem variar de pequenas condições controláveis, tais como náusea e diarreia, condições que ameaçam a vida mais. Uma vez que a maioria dos fármacos anti-cancerígenos são tóxicos, não-exposição selectiva destes fármacos ao tecido normal, inevitavelmente, provoca toxicidade. Por conseguinte, existe uma grande necessidade de uma abordagem terapêutica que possa entregar drogas selectivamente em células cancerosas. Um outro desafio com a administração de fármacos anti-cancro é a sua fraca solubilidade em água. Normalmente, os agentes de solubilização são necessários para formular estes fármacos pouco solúveis. No entanto, a maioria dos agentes de solubilização, tais como o sulfóxido de dimetilo (DMSO), Cremophor EL, e polissorbato 80 (Tween 80) podem causar toxicidade hepática e renal, hemólise, as reacções de hipersensibilidade aguda e neuropatias periféricas. 1 Assim, formulações seguros e biocompatíveis são necessários para o uso clínico de máfármacos anti-cancerígenos ly solúveis. Nanocarriers são promissores sistemas de entrega de drogas para enfrentar os desafios acima. Estes nanocarriers incluem lipossomas, 2 nanopartículas, 3 micelas, 4-7 conjugados de polímero-droga, 8 e materiais inorgânicos. 9 Vários produtos nanomedicina (por exemplo, DOXIL, Abraxane e Genexol) foram aprovados pelas agências reguladoras para tratar pacientes com câncer. 10 As micelas poliméricas são promissores transportadores de distribuição de drogas de nano-escala, que têm sido utilizados com êxito para a administração de fármacos anti-cancro. 4-7,11,12 micelas poliméricas típicas são preparadas a partir de polímeros anfifílicos através de um processo de auto-montagem. Os core-shell micelas poliméricas estruturadas incluem uma camada hidrófila e um núcleo hidrofóbico. A camada hidrófila pode estabilizar estereoquimicamente micelas e prolongar a sua circulação na corrente sanguínea. O núcleo hidrofóbico pode encapsular eficazmente d hidrofóbicotapetes. Devido ao pequeno tamanho de micelas (normalmente inferiores a 200 nM) e propriedades de longa circulação, micelas poliméricas são acreditados para atingir tumores direccionamento por meio de efeitos de retenção (EPR) (tumor passivo segmentação) e permeabilidade aumentada. A carga de droga a estabilidade é crítica para a capacidade de direccionamento tumoral de micelas. Para alcançar segmentação tumor ideal, micelas deve ter vazamento de drogas mínima antes de chegar ao local do tumor, mas liberte rapidamente a droga depois de entrar células cancerosas. Além disso, a estabilidade da formulação é também um requisito essencial para o desenvolvimento do produto, porque a estabilidade da formulação determina a viabilidade do desenvolvimento do produto, bem como a vida de prateleira de produtos desenvolvidos. Recentemente, muitos esforços têm sido feitos para melhorar o carregamento de medicamentos em transportadores de entrega. A abordagem de pró-fármaco lipofílico é uma estratégia que tem sido explorado para melhorar a carga de fármaco em nanopartículas de lípidos e emulsões. 13,14 O conjugation de lipídios com drogas pode melhorar significativamente a sua lipofilicidade e aumentar a carga e retenção nos componentes lipofílicos de nanocarriers. Aqui, descrevemos um protocolo para a preparação de doxorrubicina lipofílico pró-drogas micelas carregadas. Em primeiro lugar, é descrito o procedimento de síntese para a doxorubicina lipofílico pró-droga. Então, um protocolo para a geração de micelas com um método de película-dispersão é introduzida. Este método tem sido utilizado com sucesso nos estudos anteriores. 5 DSPE-PEG foi seleccionado como o material transportador para a preparação de micelas porque tem sido utilizado com sucesso para a entrega de drogas micela 15,16. Finalmente, descreve-se vários testes in vitro utilizados para caracterizar micela formulações e para avaliar a atividade anticâncer.

<table border="0" cellpadding="0" cellspacing="0" width="693">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">DSPE-PEG2K</td>
			<td style="width:191px;">Cordenpharm</td>
			<td style="width:119px;">LP-R4-039</td>
			<td style="width:167px;">&#62;95%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Doxorubicin</td>
			<td style="width:191px;">LC Laboratories</td>
			<td style="width:119px;">D-4000</td>
			<td>&#62;99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Palmitic Acid Hydrazide</td>
			<td style="width:191px;">TCI AMERICA&#160;&#160;</td>
			<td style="width:119px;">P000425G</td>
			<td>&#62;98.0%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:191px;">ACROS Organics</td>
			<td style="width:119px;">610981000</td>
			<td>Anhydrous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methylene chloride&#160;</td>
			<td style="width:191px;">FISHER&#160;</td>
			<td style="width:119px;">D151-4</td>
			<td>99.90%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methyl sulfoxide-d6</td>
			<td style="width:191px;">ACROS Organics</td>
			<td style="width:119px;">AC320760075</td>
			<td>NMR solvent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trifluoroacetic Acid&#160;</td>
			<td style="width:191px;">ACROS Organics</td>
			<td style="width:119px;">AC293811000</td>
			<td>99.50%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Silica Gel</td>
			<td style="width:191px;">FISHER&#160;</td>
			<td style="width:119px;">L-7446</td>
			<td>230-400 mesh</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BAKER FLEX TLC PLATES&#160;</td>
			<td style="width:191px;">FISHER&#160;</td>
			<td style="width:119px;">NC9990129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DPBS</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DU 145&#160; Prostate Cancer Cells</td>
			<td style="width:191px;">ATCC</td>
			<td style="width:119px;">HTB-81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MTT</td>
			<td style="width:191px;">ACROS Organics</td>
			<td style="width:119px;">158990050</td>
			<td>98%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 Medium</td>
			<td style="width:191px;">MEDIATECH INC&#160;</td>
			<td style="width:119px;">10041CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antibiotic-Antimycotic&#160;</td>
			<td style="width:191px;">LIFE TECHNOLOGIES&#160;</td>
			<td style="width:119px;">15240062</td>
			<td>100x stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:191px;">LIFE TECHNOLOGIES&#160;</td>
			<td style="width:119px;">10437077</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nuclear Magnetic Resonance Spectroscopy</td>
			<td style="width:191px;">Varian, Inc</td>
			<td style="width:119px;"></td>
			<td>300 NMR&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B&#252;chi R-3 Rotavapor</td>
			<td style="width:191px;">Buchi</td>
			<td style="width:119px;">1103022V1&#160;</td>
			<td>Rotary evaporator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultrasonic Bath</td>
			<td style="width:191px;">BRANSON ULTRASONICS CORPORATION&#160;</td>
			<td style="width:119px;">CPX952318R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV-VIS spectrometer Biomate 3</td>
			<td style="width:191px;">Thermo Spectronic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Zetasizer Nano ZS90&#160;</td>
			<td style="width:191px;">Malvern Instruments</td>
			<td style="width:119px;"></td>
			<td>Particle Size Analyer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microplate Spectrophotometer&#160;</td>
			<td style="width:191px;">Rio-Rad</td>
			<td style="width:119px;">Benchmark Plus&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell Culture Incubator</td>
			<td style="width:191px;">Napco</td>
			<td style="width:119px;">CO2 6000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Biological Safety Cabinet</td>
			<td style="width:191px;">Nuaire</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SigmaPlot&#160;</td>
			<td style="width:191px;">Systat Software, Inc.</td>
			<td style="width:119px;"></td>
			<td>Analytical Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-Well Cell Culture Plate</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">353072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin&#160; 0.25%</td>
			<td style="width:191px;">Corning Cellgro</td>
			<td style="width:119px;">25-053-CI</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation and characterization of lipophilic doxorubicin pro-drug micelles

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous environment through self-assembly. The hydrophobic core of micelles functions as a drug reservoir and encapsulates hydrophobic drugs. The hydrophilic shell prevents the aggregation of micelles and also prolongs their systemic circulation in vivo. In this protocol, we describe a method to synthesize a doxorubicin lipophilic pro-drug, doxorubicin-palmitic acid (DOX-PA), which will enhance drug loading into micelles. A pH-sensitive hydrazone linker was used to conjugate doxorubicin with the lipid, which facilitates the release of free doxorubicin inside cancer cells. Synthesized DOX-PA was purified with a silica gel column using dichloromethane/methanol as the eluent. Purified DOX-PA was analyzed with thin layer chromatography (TLC) and 1H-Nuclear Magnetic Resonance Spectroscopy (1H-NMR). A film dispersion method was used to prepare DOX-PA loaded DSPE-PEG micelles. In addition, several methods for characterizing micelle formulations are described, including determination of DOX-PA concentration and encapsulation efficiency, measurement of particle size and distribution, and assessment of in vitro anticancer activities. This protocol provides useful information regarding the preparation and characterization of drug-loaded micelles and thus will facilitate the research and development of novel micelle-based cancer nanomedicines.

A Figura 1 mostra o esquema de síntese de DOX-PA. DOX-PA foi sintetizada por conjugação de ácido palmítico com doxorrubicina através de uma ligação de hidrazona sensível a pH. Um ligeiro excesso de hidrazida de ácido palmítico foi usada para facilitar a finalização da reacção. Este método de reacção tem uma eficiência muito elevada e apenas uma pequena quantidade de doxorubicina permaneceu após uma reacção de 18 h (Figura 2). O rend...

Watch this Scientific Journal Video about Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles at JoVE.com

Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

Um protocolo para a preparação e caracterização de doxorrubicina lipofílico pró-fármaco carregado 1,2-distearoyl--sn-glicero-3-phosphoethanolamine- N - [amino (polietileno glicol) -2000] (DSPE-PEG) micelas é descrito.

Preparação e caracterização de Lipofílica Doxorrubicina micelas Pro-drogas

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous ...

preparation-characterization-lipophilic-doxorubicin-pro-drug

Research

JoVE Journal

Bioengineering

14.8K visualizações.  Hampton University.  Um protocolo para a preparação e caracterização de doxorrubicina lipofílico pró-fármaco carregado 1,2-distearoyl--sn-glicero-3-phosphoethanolamine- N - [amino (polietileno glicol) -2000] (DSPE-PEG) micelas é descrito. 

Vídeo: Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

Construção e Caracterização de um biorreator Fold Novel Vocal

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

Bioengenharia

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

Preparação e caracterização de novas nanopartículas simuladoras de HDL para encapsulação de fatores de crescimento nervoso

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. Atherosclerosis is also the leading cause of sudden death and myocardial infarction, instigated by unstable plaques that rupture and occlude the blood vessel without warning. Current imaging modalities cannot differentiate between stable and unstable plaques that rupture. Peptide amphiphiles micelles (PAMs) can overcome this drawback as they can be modified with a variety of targeting moieties that bind specifically to diseased tissue. Monocytes have been shown to be early markers of atherosclerosis, while large accumulation of monocytes is associated with plaques prone to rupture. Hence, nanoparticles that can target monocytes can be used to discriminate different stages of atherosclerosis. To that end, here, we describe a protocol for the preparation of monocyte-targeting PAMs (monocyte chemoattractant protein-1 (MCP-1) PAMs). MCP-1 PAMs are self-assembled through synthesis under mild conditions to form nanoparticles of 15 nm in diameter with near neutral surface charge. In vitro, PAMs were found to be biocompatible and had a high binding affinity for monocytes. The methods described herein show promise for a wide range of applications in atherosclerosis as well as other inflammatory diseases.

This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes.

Síntese de monócitos-direcionamento do Peptide Amphiphile micelas para a imagem latente da aterosclerose

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

Fabricação e caracterização de espectros ópticos de tecido contendo Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Preparação, purificação e utilização de lipossomas contendo ácido graxo

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Deteção da endotoxina em Nano-formulações usando Limulus Amoebocyte Lysate (LAL) ensaios

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Crescimento e caracterização de organóides irradiados de glândulas mamárias

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

Preparação e caracterização de Nanolipossomas para a aprisionamento de proteínas globulares hidrofílicas bioativas

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of sophisticated microelectrodes with greater resolution and expanded capabilities. The progress in fabrication technology has supported the development of biomimetic electrodes, which aim to seamlessly integrate into the brain parenchyma, reduce the neuroinflammatory response observed after electrode insertion and improve the quality and longevity of electrophysiological recordings. Here we describe a protocol to employ a biomimetic approach recently classified as nano-architecture. The use of focused ion beam lithography (FIB) was utilized in this protocol to etch specific nano-architecture features into the surface of non-functional and functional single shank intracortical microelectrodes. Etching nano-architectures into the electrode surface indicated possible improvements of biocompatibility and functionality of the implanted device. One of the benefits of using FIB is the ability to etch on manufactured devices, as opposed to during the fabrication of the device, facilitating boundless possibilities to modify numerous medical devices post-manufacturing. The protocol presented herein can be optimized for various material types, nano-architecture features, and types of devices. Augmenting the surface of implanted medical devices can improve the device performance and integration into the tissue.

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

Litografia de feixe de íons focada para etch nano-arquiteturas em microeletrodos

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of ...

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.

Three-dimensional (3D) cellular systems are relevant models for investigating organogenesis. A hydrogel-based method for biliary cysts production and their characterization is proposed. This protocol unravels the barriers of 3D characterization, with a straightforward and reliable method to assess cyst formation efficiency, sizes, and to test their functionality.

Geração e Caracterização Quantitativa de Cistos Epiteliais Biliar funcionais e polarizados

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the ...