Name: nanosensors to detect protease activity in vivo for noninvasive diagnostics
Uploaded: 2018-07-16T14:30:00
Description: Watch this Scientific Journal Video about Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics at JoVE.com

Este trabalho foi financiado por um diretor do NIH New Innovator Award (prêmio n. º DP2HD091793). Q.D.M. é suportado pelo NSF Research Bolsas de graduação (Grant no. DGE-1650044). B.A.H é suportado pelo nacional institutos de saúde GT BioMAT formação concessão sob número do prêmio 5T32EB006343, bem como do Presidente da Geórgia Tech Fellowship. G.A.K. detém um prêmio de carreira na Interface científica do fundo de boas-vindas de Burroughs. O conteúdo é exclusivamente da responsabilidade dos autores e não representa necessariamente a opinião oficial do institutos nacionais da saúde.

Aprovação institucional do cuidado institucional do Animal e Comissão de utilização (IACUC) na instituição do pesquisador é necessária efectuar as seguintes experiências com animais. Além disso, instalações de cuidados de animais padrão (por exemplo, câmaras, capas de animais esterilizadas, isofluorano câmaras para anesthetization e CO2 câmaras para eutanásia de ponto de extremidade ética de habitação) são necessárias efectuar correctamente estas experimentos. Formação especia...

<ol>
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Este método descreve o desenvolvimento de actividades nanosensors consistindo de substratos de protease, conjugados com um núcleo de nanopartículas. O evento de clivagem proteolítica é apelidado do "interruptor farmacocinético", porque produtos clivados peptídeo são menores do que o limite de tamanho renal de filtragem de 5 nm23 e filtro na urina para produzir um sinal não-invasivo. Portanto, é importante usar nanopartículas ou transportadoras com um raio hidrodinâmico maior que 5 nm, ...

Proteases são enzimas multi-funcional que especializa-se na hidrólise das ligações peptídicas e tem um controlo significativo sobre muitos processos biológicos, incluindo a homeostase, allostasis e doença1. Um estado alterado de atividade de protease tem sido correlacionado a uma variedade de doenças, incluindo câncer e doenças cardiovasculares, tornando atraente candidatos de proteases para desenvolvimento em biomarcadores clínicos2,3. Além disso, a atividade de protease é pathogeneses funcionalmente vinculados a distintos, os resultados dos pacientes e prognóstico da doença4. Amplamente, biosensores foram desenvolvidos para detectar vários fenômenos biológicos e doenças, como câncer, doenças neurodegenerativas e transferência de elétrons processa5,6,7,8 , 9. mais especificamente, sensores de protease baseada no substrato foram desenvolvidos para detectar atividade de protease e incluem fluorogenic sondas para diagnóstico por imagem10 e desvendar rotulados substratos de peptídeo para in vitro deteção por espectrometria de massa11. Além disso, baseado na atividade sondas têm sido desenvolvidas, que contêm o substrato, como regiões que ligam ou modificar o destino protease12. Com este método, a protease alvo é irreversivelmente inibida quando o sítio ativo é modificado, e análise requer a colheita de tecidos, que limita em vivo aplicações. No entanto, é importante sentir protease atividade na vivo, porque a regulação da atividade de protease é fortemente dependente do contexto de outras atividades biológicas, tais como a presença de inibidores endógenos.
O objetivo deste trabalho é descrever a formulação de nanosensors actividades que detectam protease atividade na vivo , produzindo um sinal mensurável na urina. Esta plataforma é usada como um diagnóstico não-invasivo para discriminar doenças complexas como câncer, usando a atividade de protease de desregulação como biomarcador funcional. Nossa plataforma de nanosensor consiste em nanopartículas de óxido de ferro (IONP) conjugadas com substratos da protease. Estes substratos são terminados por um repórter fluorescente que é liberado quando proteases fendem o substrato. Estes IONPs circulam na vivo, localizar sites de doença e expõem os substratos de proteases associada a doença ativas. Após a clivagem, repórteres fluorescentes são liberados e, devido ao seu pequeno tamanho, são filtrados na urina, enquanto os substratos uncleaved sobre a IONP permanecem no corpo. Portanto, um aumento em protease atividades na vivo resultará em concentrações mais altas de repórter na urina (Figura 1). Desde que nossa plataforma é um teste de urina, sem plataforma de imagem é necessária e sinais de diagnóstico são enriquecidos na urina.
Esta plataforma pode ser projetada para detectar uma variedade de doenças, incluindo câncer, fibrose e trombose13,14. Aqui descrevemos o projeto de nanosensors para detectar elevações em Matrix metallopeptidase 9 (MMP9) atividade como um biomarcador de câncer colorretal. O câncer colorretal é a segunda causa principal de morte por cancro nos Estados Unidos, com uma estimativa 136.800 novos casos e 50.300 mortes em 2014 só15. Células de tumor colorretal produzem MMP9, que foi mostrado para conduzir a progressão maligna, degradação de matriz, bem como metástase16. Além disso, nós identificamos um substrato adequado do peptide (PLGVRGK) para MMP9 da literatura17. Esta plataforma pode ser utilizada para detecção precoce de câncer e de baixo custo point-of-care diagnósticos13,14,18,19,20,21.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57937/57937fig1.jpg"> Figura 1: esquemático da Nanosensor atividade In vivo. Nanosensors circular através do corpo e localizar a locais de doença. Em seguida, doença relacionada com proteases cleave substratos de peptídeo apresentados pela IONPs. O tamanho dos fragmentos clivados permite a compensação renal, levando-os a localizar na urina. Depois que o animal urina, esses fragmentos de peptídeo podem ser analisados por sua molécula repórter. <a href="https://www.jove.com/files/ftp_upload/57937/57937fig1large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>

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			<td height="21" style="height:21px;width:375px;">0.2 &#181;m syringe filters</td>
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			<td height="21" style="height:21px;width:375px;">15 mL conicals</td>
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			<td height="21" style="height:21px;width:375px;">250 mL Erlenmeyer flask</td>
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			<td style="width:233px;">89000-362</td>
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			<td height="21" style="height:21px;width:375px;">Glacial acetic acid</td>
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			<td style="width:233px;">97064-482</td>
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			<td height="21" style="height:21px;width:375px;">Albumin from Bovine Serum (BSA)</td>
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			<td height="21" style="height:21px;width:375px;">Iron (III) chloride hexahydrate</td>
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			<td height="21" style="height:21px;width:375px;">Iron (II) chloride tetrahydrate</td>
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			<td height="21" style="height:21px;width:375px;">Epichlorohydrin</td>
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			<td height="21" style="height:21px;width:375px;">DMF</td>
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			<td height="21" style="height:21px;width:375px;">Ammonium Hydroxide</td>
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			<td height="21" style="height:21px;width:375px;">Tris-HCl</td>
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			<td height="21" style="height:21px;width:375px;">Tris base</td>
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			<td height="21" style="height:21px;width:375px;">PBS tablets</td>
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			<td height="21" style="height:21px;width:375px;">Amicon 15 mL 10k filters, 24 pk</td>
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			<td height="21" style="height:21px;width:375px;">Amicon 15 mL 100k filters, 24 pk</td>
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			<td height="21" style="height:21px;width:375px;">Zetasizer Nano ZS</td>
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nanosensors to detect protease activity in vivo for noninvasive diagnostics

Proteases são enzimas multi-funcional que especializa-se na hidrólise das ligações peptídicas e controlam grandes processos biológicos incluindo a homeostase e allostasis. Além disso, a atividade de protease de desregulação drives patogênese e é um biomarcador funcional de doenças como o câncer; Portanto, a capacidade de detectar protease atividade na vivo pode fornecer informações clinicamente relevantes para diagnósticos biomédicos. O objetivo do presente protocolo é criar nanosensors que sondam protease atividade na vivo , produzindo um sinal quantificável na urina. Estes nanosensors da protease consistem em dois componentes: uma de nanopartículas e substrato. As funções de nanopartículas para aumentar a circulação Half-Life e substrato entrega doença nos sites de destino. O substrato é uma sequência do peptídeo curto (6-8 AA), que é projetada para ser específico para uma protease alvo ou grupo de proteases. O substrato é conjugado com a superfície da nanopartículas e é terminado por um repórter, como um marcador fluorescente, para a deteção. Como desregulação proteases fendem o substrato de peptídeo, o repórter é filtrado na urina para quantificação como um biomarcador de atividade de protease. Neste documento descrevemos a construção de um nanosensor para matriz metaloproteinase 9 (MMP9), que está associado a progressão do tumor e metástases, para detecção do câncer colorretal em um modelo do rato.

A maioria da população das IONPs é em torno do diâmetro médio, que varia de 40 a 50 nm. Após pegylation, nesta faixa de tamanho tem uma meia-vida de circulação de aproximadamente 6 horas13 na vivo (ver Figura 2a). Se alguém quiser selecionar para uma série de determinado tamanho, um pode usar cromatografia de exclusão para isolar frações IONP com diâmetros diferentes. As nanopartículas pela TEM aparecerá como ...

Watch this Scientific Journal Video about Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics at JoVE.com

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases são rigidamente regulamentadas enzimas envolvidas em processos biológicos fundamentais e desregulação protease atividade unidades de progressão de doenças complexas como câncer. O objetivo do método é criar nanosensors que medem protease atividade na vivo , produzindo um sinal de clivagem que é detectável de urina de anfitrião e discrimina a doença.

Nanosensors para detectar Protease atividade In Vivo para diagnóstico não-invasivo

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

This method can help answer key questions in protea mix and diagnostics, such as how to design probes that can detect the progression of diseases by measuring proteates'activity in vivo. The main advantage of this method is that the diagnositics developed will be noninvasive, and can actually amplify the detection of biological signals. This method can be applied to numerous diseases, because many proteuses are actually major drivers of pathogenesis.In the chemical fume hood, transfer one ml of 30%ammonium hydroxide to a 15 ml conical tube. Place this tube into an ice bucket and let it chill for 30 minutes. Wash a 250 ml Ehrlenmeyer flask with deionized water.Then add 10 ml of double distilled water. Submerge the flask in an ice bath resting on a heat plate. Using a plastic pipette, begin the flow of nitrogen gas into the double distilled water in the flask.Bubble for 15 minutes to deoxygenate. Next, weigh out 4.5 g of dextrine in a 15 ml conical tube. Add water such that the final volume of the mixture including the dextrine is 20 ml.Vortex vigorously to dissolve the dextrine. Weigh out 78.5 mg of iron 3 chloride hexahydrate and add this to the dextrine solution. Vortex to dissolve.Using a 0.2 um filter, filter the resulting solution. After this, transfer 9 ml of the deoxygenated water to the 15 ml conical tube containing the 1 ml of chilled ammonium hydroxide. Return the conical tube to the ice bucket to chill.Weigh out 73 mg of iron 2 chloride tetrahydrate and dissolve it in the remaining 1 ml of deoxygenated double distilled water. Use a 0.2 um filter to filter the solution. Then, transfer the dextrine iron 3 solution into the 250 ml Ehrlenmeyer flask.Deoxygenate with nitrogen gas for 15 minutes on ice, while mixing at 1, 600 rpm. Cap the flask with a rubber septum. Using an 18 gauge needle, puncture the septum to flow nitrogen gas into the flask.Insert a separate 18 gauge needle, which will be the flow outlet. Next, use a 1 ml syringe to add 467 ul of the iron 2 solution to the dextrine iron 3 solution. Stir at 1, 600 rpm.Add the chilled dilute ammonium hydroxide solution dropwise to initiate the nucleation process. Make sure each drop mixes well before adding another. Stop the flow of nitrogen gas and remove the rubber septum.Remove the ice bath and replace it with a bath of warm water. Heat the solution to 75 degrees Celsius and then incubate for 75 minutes. Remove the flask from the hot plate.Doubly filter the solution through a 0.2 um filter and then a 0.1 um filter to remove any coarse particles. Using 100 kDa molecular weight cutoff concentrators, buffer exchange the particles into double distilled water to remove excess dextrine. If the flow through remains dark after two to three spins, replace the filters as they may have broken.Next, resuspend the iron oxide nanoparticles in double distilled water at a concentration of 10 mg per ml. Add 1.6 volumes of 5 molar sodium hydroxide, then add 0.65 volumes of epichlorohydrine. Using a plate shaker, rigorously mix at room temperature for 12 hours.Use a 20 ml syringe with an 18 gauge needle to transfer the iron oxide nanoparticle solution into a dialysis membrane with a 50 kDa molecular weight cutoff. Place the dialysis membrane into 4 liters of double distilled water. Incubate at room temperature overnight.After determining the concentration, bring the iron oxide nanoparticles to a concentration of 5 mg per ml. Add ammonium hydroxide to reach 20%and shake at room temperature for at least 12 hours to amenate the surface of the iron oxide nanoparticles. Then, buffer exchange using 30 kDa molecular weight cutoff filters.Use dynamic light scattering to determine the hydrodynamic radius of the iron oxide nanoparticles as outlined in the text protocol. After synthesizing a peptide substrate, aliquot 0.5 mg of iron oxide nanoparticles into the 10 kDa molecular weight cutoff spin filter tube. Buffer exchange into coupling buffer using a 10 kDa molecular weight cutoff spin filter as outlined in the text protocol.Dissolve SIA in DMF to reach a concentration of approximately 30 mg per ml. Then, add SIA to the iron oxide nanoparticles at a mole ratio of 500 to one. After incubation and buffer exchange, bring the final product to a concentration of 1 mg per ml.Mix the peptide of interest with 20 kDa thiol terminated polyethylene glycol, and then mix this peptide glycol solution with iron oxide nanoparticles. Cover the tubes in foil to prevent photo bleaching the fluorescent molecules. And incubate overnight at room temperature on a plate shaker set the to the highest speed.Next, add L cystine at a molar ratio of 500 to one to the iron oxide nanoparticles. Incubate for one hour at room temperature on a shaker set to the highest speed. After this, make an 18 ul nanoparticle solution using PVS, and a 200 nanomolar concentration of peptide.Add 2 ul of the protease of interest. Incubate in a plate reader for one hour at 37 degrees Celsius taking flourescence measurements every one to two minutes to monitor cleavage. Immediately after injecting the prepared nanosensor solution via tail vein injection, place the mice into metabolic cages, and note the time of injection for each mouse.Combine 2 ul of urine with 5 ul of prepared magnetic beads. Using PVS, bring the final total volume to 50 ul. Incubate at room temperature for 60 minutes.Next, wash twice using 50 ul of PVS each time, while using a magnetic separator to collect the magnetic beads after each wash. Elute twice with 32.5 ul of 5%glacial acetic acid. Use 35 ul of 2 molar tris to neutralize the pool dilution and achieve a final ph around 7.After this, use a plate reader at appropriate excitation and emission wavelengths to quantify the urine flourescence. Calculate the concentration of fluorescent reporter against a ladder of known concentrations of free flourophor. In this study, activity based nanosensors are developed by conjugating protease substrates to a nanoparticle core.The average diameter of iron oxide nanoparticles is observed to be between 40 and 50 nm. After pecolation, the circulation half life for this size range is seen to be approximately 6 hours. After successful peptide conjugation, spectral absorbance analysis reveals a distinct absorbance peak at the maximum excitation wavelength of the fluorescent reporter.The ability of the probe to sense protease activity is then tested by incubating an aliquot of nanosensors with recombinant proteases. It is typical to observe substrate cleavage velocities that follow Michaelis-Menten kinetics. Nanosensors coated with a near infrared fluorescent reporter are then used to visualize the pharmacokinetics in mouse models of cancer by whole animal fluorescent imaging.A fluorescent signal is seen to localize in the urine of mice, bearing LS 174 t colorectal, 60 to 90 minutes after intravenous administration of the nanosensors. Significantly lower fluorescent signals are observed in the bladders of control mice. It should be noted that the signal observed in the liver arises because particles are eventually scavenged by monocites and and macrophages in the ridiculoendophilial system, which are commonly found in the liver, spleen, and lymph nodes.Once mastered, this technique can be done in four days, if it's performed properly. While attempting this procedure, it's important to remember that flourescence is the primary readout, so it's important to keep the peptide substrates with fluorescent markers in the dark. Following this procedure, other methods like biodistribution studies and in viva imaging can be performed in order to answer additional questions, like localization of particles to various organs.After its development, this technique paved the way for researchers in the field of diagnostics to explore cancer biomarkers in mice. After watching this video, you should have a good understanding of how to synthesize and characterize iron oxide nanoparticles, use these to build nanosensors, then use the nanosensors in vivo. Don't forget that working with the chemicals involved in a nanoparticle synthesis can be extremely hazardous, and precautions such as personal protective equipment and working within a fume hood should always be taken while performing this procedure.

Research

JoVE Journal

Bioengineering

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Projeto de um biorreator de pressão cíclica para o Ex Vivo Estudo de válvulas cardíacas Aórtica

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Bioengenharia

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Quantitative FRET F&#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

Quantitativa FRET (Förster Resonance Energy Transfer) Análise para SENP1 Determinação Cinética Protease

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

FIBS-enabled Noninvasive Metabolic Profiling

FIBS habilitado Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

A pele completa Defeito Modelo Estudar a vascularização de Biomateriais<em&gt; In Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

Levitação magnética Juntamente com imagem Portátil e Análise para a doença Diagnostics

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Desenvolvimento de um Biosensor eletroquímico do DNA para detectar um patógeno de origem alimentar

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Calorespirometry: Uma abordagem poderosa, não-invasiva para investigar o metabolismo energético celular

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

Rígida incorporação de fixo e manchado, inteiro, espécimes de milímetro-escala para histologia seção livre 3D por Microtomografia computadorizada

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics

Methylation Specific Multiplex Droplet PCR usando dispositivo gerador de gotícula de polímero para diagnósticos hematológicos

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

Desenvolvimento de tecido cardíaco humano organizado 3D dentro de uma plataforma microfluida

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...