Name: an aptamer-based sensor for unchelated gadolinium(iii)
Uploaded: 2017-01-09T15:00:00.000+00:00
Duration: 5 min 15 s
Description: Watch this Scientific Journal Video about An Aptamer-based Sensor for Unchelated GadoliniumIII at JoVE.com

We would like to gratefully acknowledge Dr. Milan N Stojanovic from Columbia University, New York, NY for valuable scientific input. This work is supported by funding from the California State University East Bay (CSUEB) and the CSUEB Faculty Support Grant-Individual Researcher. O.E., T.C., and A.L. were supported by the CSUEB Center for Student Research (CSR) Fellowship.

NOTA: l&#39;acqua di biologia molecolare di grado è utilizzato in tutte le preparazioni tampone ed una soluzione. Tutti i tubi monouso (microcentrifuga e PCR) e puntali sono DNase- e RNase-free. Si prega di consultare la scheda di sicurezza (SDS) per tutte le sostanze chimiche prima dell&#39;uso. L&#39;utilizzo di adeguati dispositivi di protezione individuale (DPI) è fortemente raccomandato. 1. Preparazione dei aptamero archivio Solutions <ol><li> Acquisto 2 basi di polydeoxynucleotide commercio. Ordinare entrambi i filamenti con purificazione tramite cromatografia liquida ad alte prestazioni (HPLC). Parte 1 (Gd-aptamero): 5 &#39;- / 56-FAM / AGGCTCTCGGGACGACCAGTTGGTCCCGCTTTATGTGTCCCGAG-3&#39; Parte 2 (QS): 5&#39;-GTCCCGAGAGCCT / 3Dab / -3 &#39; </li><li> Sciogliere ogni filo in acqua per fare 100 micron singole soluzioni di riserva del Gd-aptamer e il QS. </li><li> Conservare queste soluzioni a -20 ° C. Le soluzioni sono stabili finora, per 3 anni. </li><li> Per ridurre al minimo freezcicli e-disgelo, memorizzare le soluzioni madre in 10 microlitri aliquote. </li></ol> 2. Preparazione del 2x Gd-sensore Solution <ol><li> Preparare il tampone (20 mM HEPES, 2 mM MgCl 2, 150 mM NaCl, 5 mM KCl). Regolare il pH a ~ 7,4 con NaOH e HCl, e filtrare bottiglia migliori filtri sterili monouso con 0,2 micron membrana PES. Conservare in bottiglie sterili. Se filtrato e conservato correttamente (a temperatura ambiente), il buffer è stabile finora, per 2 anni. </li><li> Diluire 1 ml di soluzione di Gd-aptamer magazzino (dal punto 1.2) e 2 ml di soluzione di riserva QS (dal punto 1.2) in 497 ml di tampone. Mescolare bene con un vortice. La loro concentrazione nella soluzione Gd-sensore 2x sono 200 e 400 nm, rispettivamente. NOTA: Il volume della soluzione Gd-sensore 2x preparato in questa fase è 500 ml, che è sufficiente per testare 6 - 7 concentrazioni variabili di Gd 3+ per la curva di calibrazione e le soluzioni di contrasto(Fase 3). Ogni campione darà pozzetti duplicati in una piastra da 384 pozzetti. <ol><li> Regolare il volume della soluzione di Gd-sensore 2x in base al numero di soluzioni Gd 3+ che deve essere testato. </li></ol></li><li> Trasferire la soluzione Gd-sensore 2x in 9 provette PCR, con 50 microlitri in ogni provetta. Mettere le provette in un termociclatore. </li><li> Impostare il programma nel termociclatore per riscaldare la soluzione nei tubi a 95 ° C, tenere premuto per 5 min e quindi raffreddare lentamente la soluzione a 25 ° C sopra ~ 15 min (al tasso di ~ 0.05 - 0,1 ° C / S). Il ciclo di riscaldamento e raffreddamento è di garantire ibridazione ottimale tra il Gd-aptamer e QS. risultati parziali ibridazione in tempra incompleta e uno sfondo più elevata fluorescenza del sensore. Se un termociclatore non è disponibile, effettuare questo processo utilizzando un bagno di acqua calda, invece. </li><li> Una volta raffreddata a 25 ° C, utilizzare immediatamente la soluzione, o mantenere nel termociclatore (fino a circa 2 h) fino al momento di essereusato. Quando un bagno di acqua viene utilizzata per il riscaldamento, lasciare le provette nel bagno come l&#39;acqua si raffredda lentamente a temperatura ambiente. </li></ol> 3. costruire la curva di fluorescenza taratura e rilevare la presenza di Unchelated Gd 3+ in una soluzione di Gd Agente Contrast <ol><li> Sciogliere GdCl 3 solido nel tampone (lo stesso buffer come al punto 2.1). </li><li> Attraverso diluizione in serie, preparare 100 ml ciascuna di 6 diverse soluzioni Gd 3+ in tubi microcentrifuga al doppio delle concentrazioni finali desiderati per la curva di calibrazione (2x soluzioni). <ol><li> Ad esempio, per costruire una calibrazione per 0 (tampone solo senza GdCl 3), 50, 100, 200, 400, e 800 nM di Gd 3+, preparare soluzioni contenenti 0, 100, 200, 400, 800, e 1600 nM dello ione. Assicurati di includere sempre il &#39;vuoto&#39; con 0 ns Gd 3+ come il controllo negativo. </li></ol></li><li> Sciogliere il mezzo di contrasto per essere provaDE al tampone. Preparare 2 o 3 diverse concentrazioni delle soluzioni di contrasto tramite diluizione in serie. NOTA: Test 3 diverse concentrazioni della soluzione di agente di contrasto è raccomandato. Questo per garantire che queste concentrazioni sono all&#39;interno del range lineare. Se i campioni esaminati non presentano un rapporto lineare, ridurre le concentrazioni di agente di contrasto utilizzato. </li><li> Prendere le provette PCR contenente la soluzione 2x Gd-sensore dal punto 2.5 del termociclatore. </li><li> Aggiungere 50 ml di ciascuna soluzione Gd 3+ dal punto 3.2 in 6 delle provette PCR 9 contenente la soluzione 2x Gd-sensore. Mescolare pipettando su e giù. Ogni tubo PCR contiene ora la concentrazione desiderata di Gd 3+ da testare, 100 nM Gd-aptamer, e 200 nM QS. </li><li> Per le provette PCR restanti contenenti la soluzione Gd-sensore 2x, aggiungere 50 ml delle soluzioni di contrasto dal punto 3.3. Mescolare bene pipettando su e giù un paio di volte. </li><li> Incubare le solutions nei tubi PCR per circa 5 min a temperatura ambiente. Essi possono essere lasciati riposare per un massimo di 30 min. </li><li> Trasferire 45 ml di ciascun tubo in una piastra da 384 pozzetti. Ogni tubo di PCR darà pozzetti in duplicato. </li><li> Registrare la fluorescenza di ciascun pozzetto su una piastra-reader. Il fluoroforo (FAM) utilizzati per la progettazione Gd-sensore è di eccitazione e di emissione massimi di 495 e 520 nm, rispettivamente, come indicato sul sito web del fornitore. Scegliere opportune lunghezze d&#39;onda di eccitazione e di emissione o filtri a seconda che la piastra-lettore è monochromator- o filter-based. </li><li> Tracciare il grafico della fluorescenza in unità di fluorescenza arbitrarie (AFU) contro la concentrazione di Gd 3+. </li><li> Tracciare il grafico come il cambiamento di fluorescenza volte contro la concentrazione di Gd 3+. Calcolare la variazione di fluorescenza volte dividendo la AFU di ciascuna concentrazione dal AFU della soluzione &#39;in bianco&#39; (con 0 nM di Gd 3+). Il cambiamento di fluorescenza piega consentirà di normalization dei risultati, qualora il display AFU alcuni periodica (diversi giorni, ecc) variazioni. </li><li> Confrontare l&#39;emissione di fluorescenza della soluzione contenente l&#39;agente di contrasto e &#39;blank&#39;, che è la soluzione contenente 0 nM GdCl 3 (tampone solo). NOTA: Una maggiore fluorescenza della soluzione GBCA implica la presenza di unchelated Gd 3+, che può richiedere ulteriore purificazione del mezzo di contrasto. La quantità di unchelated Gd 3+ attuale può essere stimata utilizzando la curva di calibrazione costruita nel punto 3.10 o 3.11. </li></ol>

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The authors declare that they have no competing financial interests.

Utilizzando il Gd-sensore aptameri base, un aumento di emissione di fluorescenza che è proporzionale alla concentrazione di unchelated Gd 3+ è osservato. Per ridurre al minimo la quantità di campione usato, il saggio può essere eseguito in 384 pozzetti micropiastra con un volume di campione totale di 45 microlitri per pozzetto. In questo disegno, la scelta di fluoresceina (FAM) e DABCYL (Dab) è basata principalmente sul costo dei reagenti; modificare la lunghezza d&#39;onda di emissione, un diverso accop...

La crescente importanza della risonanza magnetica (MRI) nella diagnosi clinica, che è limitata dalla sensibilità intrinseca della tecnica, ha portato alla rapida crescita della ricerca per lo sviluppo di nuovi agenti di contrasto a base di gadolinio (GBCAs) 1. GBCAs sono molecole che vengono somministrati per migliorare la qualità dell&#39;immagine, e che in genere hanno la struttura chimica di uno ione trivalente gadolinio (Gd 3+) coordinato ad un ligando polidentato. Questa complessazione è di importanza critica come unchelated Gd 3+ è tossico; essa è stata implicata nello sviluppo di fibrosi sistemica nefrogenica in alcuni pazienti con malattia renale o insufficienza 2. Di conseguenza, rilevando lo ione libero acquosa è fondamentale per garantire la sicurezza dei GBCAs. La presenza di unchelated Gd 3+ nelle soluzioni GBCA spesso è il risultato di una reazione incompleta tra il ligando e lo ione, la dissociazione del complesso, o displacement da altri cationi metallici biologiche 3. Tra le varie tecniche attualmente utilizzate per la determinazione della presenza di Gd 3+, quelli che usano cromatografia e / o spettrometria grado più alto in termini di versatilità e applicabilità 4. Tra i loro punti di forza sono l&#39;alta sensibilità e precisione, la capacità di analizzare diverse matrici di campioni (compresi siero umano 5, urine e capelli 6, acque di scarico 7 e contrasto agente formulazioni 8), e la quantificazione simultanea di più 3+ complessi di Gd (una scheda di studi prima al 2013 è descritto in una revisione completa da Telgmann et al.) 4. L&#39;unico inconveniente è che molti di questi metodi richiedono strumentazioni (come la spettrometria di massa al plasma accoppiato induttivamente) 4 che alcuni laboratori non possono avere accesso. Nel contesto della nuova scoperta GBCA alla ricerca e livelli proof-of-concept, arMetodo elatively più conveniente, rapida e conveniente spettroscopica basata (come l&#39;assorbimento UV-Vis o fluorescenza) può servire come una valida alternativa. Con queste applicazioni in mente, un sensore di aptameri a base acquosa fluorescente per Gd 3+ è stato sviluppato 9. Il aptameri (Gd-aptamero) è una lunga molecola di DNA a singolo filamento 44-base con una specifica sequenza di basi che è stata isolata attraverso il processo di evoluzione sistematica dei ligandi di arricchimento esponenziale (SELEX) 9. Per adattare l&#39;aptamero in un sensore fluorescente, un fluoroforo è attaccato al terminale 5 &#39;del filamento, che viene quindi ibridato con un filamento di tempra (QS) con 13 basi complementari (Figura 1). Il QS è etichettato con una molecola quencher scuro al &#39;capolinea 3. In assenza di Gd 3+, il sensore (Gd-sensore), costituito da un rapporto 1: 2 moli di Gd-aptamero e QS rispettivamente avrà emissione di fluorescenza minima dovuta ttrasferimento di energia o dal fluoroforo alla quencher. L&#39;aggiunta di soluzione acquosa di Gd 3+ sposterà QS dal Gd-aptamer, con un conseguente aumento di emissione di fluorescenza. <img alt="Figura 1" src="/files/ftp_upload/55216/55216fig1.jpg" /> Figura 1. Il sensore (Gd-sensore) che consiste nella aptamer 44-passo lungo (Gd-aptamero) codificata con fluoresceina (un fluoroforo) e il 13-passo lungo tempra filamento (QS) contrassegnati con DABCYL (un quencher scuro) . In assenza di unchelated Gd 3+, la fluorescenza del sensore è minima. Con l&#39;aggiunta di Gd 3+, spostamento del QS si verifica e si osserva un aumento di emissione di fluorescenza. <a href="http://ecsource.jove.com/files/ftp_upload/55216/55216fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> Vi è attualmente, quello comunemente usato metodo spettroscopico-based per rilevareing acquosa 3+ Gd. Questo saggio utilizza arancione xilenolo molecola, che subisce uno spostamento della lunghezza d&#39;onda massima assorbimento dai 433 ai 573 nm su di chelazione allo ione 10. Il rapporto tra queste due assorbanza massimi può essere utilizzato per quantificare la quantità di unchelated Gd 3+. Il sensore aptamero è un&#39;alternativa (può anche essere complementari) al dosaggio xilenolo arancio, come i due metodi hanno le condizioni di reazione (come il pH e la composizione delle soluzioni tampone utilizzato), selettività bersaglio, gamme lineari di quantificazione, e le modalità di rilevamento 9.

<table><tbody><tr><td>Gd-aptamer</td><td>IDTDNA</td><td>Input sequence and fluorophore modification in the order form</td><td>A fluorophore with a different emission wavelength may be used. The aptamer may also be ordered from another company.</td></tr><tr><td>Quenching strand</td><td>IDTDNA</td><td>Input sequence and quencher modification in the order form</td><td>A different quencher for optimal energy transfer from the fluorophore may be used. The aptamer may also be ordered from another company.</td></tr><tr><td>Molecular biology grade water</td><td></td><td></td><td>No specific manufacturer, both DEPC or non-DEPC treated work equally well</td></tr><tr><td>Gadolinium(III) chloride anhydrous</td><td>Strem</td><td>936416</td><td>Toxic</td></tr><tr><td>HEPES</td><td>Fisher Scientific</td><td>BP310-500</td><td></td></tr><tr><td>Magnesium chloride anhydrous</td><td>MP Biomedicals</td><td>0520984480 - 100 g</td><td></td></tr><tr><td>Sodium Chloride</td><td>Acros Organics</td><td>327300025</td><td></td></tr><tr><td>Potassium chloride</td><td>Fisher Scientific</td><td>P333-500</td><td></td></tr><tr><td>Sodium hydroxide, pellets</td><td>Fisher Scientific</td><td>BP359</td><td>Corrosive</td></tr><tr><td>Hydrochloric acid</td><td>Fisher Scientific</td><td>SA49</td><td>Toxic and corrosive</td></tr><tr><td>384-well low flange black flat bottom polystyrene NBS plates</td><td>Corning</td><td>3575</td><td>Plates which are suitable for fluorescence reading are required.</td></tr><tr><td>Nalgene Rapid-Flow sterile disposable bottle top filter</td><td>Thermo Scientific</td><td>5680020</td><td>The bottle top is fitted with 0.2 micron PES membrane</td></tr><tr><td>Disposable sterile bottles 250 mL</td><td>Corning</td><td>430281</td><td>A larger or smaller bottle may be used</td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td></td><td></td><td>No specific manufacturer, as long as they are DNAse and RNAse-free</td></tr><tr><td>0.2 mL PCR tubes</td><td></td><td></td><td>No specific manufacturer, as long as they are DNAse and RNAse-free</td></tr><tr><td>Micropipets</td><td></td><td></td><td>No specific manufacturer</td></tr><tr><td>Pipet tips (non filter) of appropriate sizes</td><td></td><td></td><td>No specific manufacturer, as long as they are DNAse and RNAse-free</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Plate reader</td><td>Biotek Synergy H1</td><td></td><td>Plate readers from other manufacturers would work equally well</td></tr></tbody></table>

an aptamer-based sensor for unchelated gadolinium(iii)

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples of gadolinium-based contrast agents as a result of incomplete reactions between the ligand and the ion, or as a dissociation product. Since the ion is toxic, its detection is of critical importance. Herein, the design and usage of an aptamer-based sensor (Gd-sensor) for Gd3+ are described. The sensor produces a fluorescence change in response to increasing concentrations of the ion, and has a limit of detection in the nanomolar range (~100 nM with a signal-to-noise ratio of 3). The assay may be run in an aqueous buffer at ambient pH (~7 - 7.4) in a 384-well microplate. The sensor is relatively unreactive toward other physiologically relevant metal ions such as sodium, potassium, and calcium ions, although it is not specific for Gd3+ over other trivalent lanthanides such as europium(III) and terbium(III). Nevertheless, the lanthanides are not commonly found in contrast agents or the biological systems, and the sensor may therefore be used to selectively determine unchelated Gd3+ in aqueous conditions.

Una tipica variazione di fluorescenza della soluzione di Gd-sensore in presenza di unchelated Gd 3+ è mostrato in Figura 2. L&#39;emissione può essere tracciata come la variazione di fluorescenza piega (Figura 2A) o la fluorescenza grezza lettura (Figura 2B) in unità arbitrarie (AFU). Entrambe le trame producono curve di calibrazione molto simili, con un intervallo lineare per le concentrazioni di Gd 3+ inferiore...

Watch this Scientific Journal Video about An Aptamer-based Sensor for Unchelated GadoliniumIII at JoVE.com

An Aptamer-based Sensor for Unchelated GadoliniumIII

The use of polydeoxynucleotide (44-mer aptamer) molecules for sensing unchelated gadolinium(III) ion in an aqueous solution is described. The presence of the ion is detected via an increase in the fluorescence emission of the sensor.

Un sensore Aptamero-based per Unchelated gadolinio (III)

A method for determining the presence of unchelated trivalent gadolinium ion (Gd3+) in aqueous solution is demonstrated. Gd3+ is often present in samples ...

an-aptamer-based-sensor-for-unchelated-gadolinium-iii

Research

JoVE Journal

Chemistry

An Aptamer-based Sensor for Unchelated Gadolinium(III)

7.2K Views.  California State University, East Bay. The use of polydeoxynucleotide (44-mer aptamer) molecules for sensing unchelated gadolinium(III) ion in an aqueous solution is described. The presence of the ion is detected via an increase in the fluorescence emission of the sensor. 

Video: An Aptamer-based Sensor for Unchelated GadoliniumIII

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

Indagini su GA (III) Complesso di EOB-DTPA e la sua<sup&gt; 68</sup&gt; Ga marcata radioattivamente analogico

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

Ricerca

Chimica

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance imaging (MRI). Their purpose is to enhance the relaxation rate of water protons in tissue, thus increasing the MR image contrast and the specificity of the MRI measurements. Current clinically approved contrast agents are low molecular weight molecules that are rapidly cleared from the body. The use of dendrimers as carriers of paramagnetic chelators can play an important role in the future development of more efficient MRI contrast agents. Specifically, the increase in local concentration of the paramagnetic species results in a higher signal contrast. Furthermore, this CA provides a longer tissue retention time due to its high molecular weight and size. Here, we demonstrate a convenient procedure for the preparation of macromolecular MRI contrast agents based on poly(amidoamine) (PAMAM) dendrimers with monomacrocyclic DOTA-type chelators (DOTA &#8211; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate). The chelating unit was appended by exploiting the reactivity of the isothiocyanate (NCS) group towards the amine surface groups of the PAMAM dendrimer to form thiourea bridges. Dendrimeric products were purified and analyzed by means of nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis. Finally, high resolution MR images were recorded and the signal contrasts obtained from the prepared dendrimeric and the commercially available monomeric agents were compared.

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

This protocol describes the preparation and characterization of a dendrimeric magnetic resonance imaging (MRI) contrast agent that carries cyclen-based macrocyclic chelates coordinating paramagnetic gadolinium ions. In a series of MRI experiments in vitro, this agent produced an amplified MRI signal when compared to the commercially available monomeric analogue.

Preparazione e<em&gt; In Vitro</em&gt; Caratterizzazione del dendrimero a base di agenti di contrasto per risonanza magnetica

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance ...

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

MR Imaging Molecolare del cancro alla prostata con un piccolo Molecular CLT1 Peptide mirato di contrasto agente

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

Medicina

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.

Biofunctionalized Prussian Blue Nanoparticelle per applicazioni di imaging multimodale molecolari

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

Bioingegneria

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect resulting in significant reductions in systemic toxicity. Nonetheless, insufficient release of encapsulated drug from liposomes has limited their clinical efficacy. Temperature-sensitive liposomes have been engineered to provide site-specific release of drug in order to overcome the problem of limited tumor drug bioavailability. Our lab has designed and developed a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, to provide triggered release of CDDP at solid tumors. Heat-activated delivery in vivo was achieved in murine models using a custom-built laser-based heating apparatus that provides a conformal heating pattern at the tumor site as confirmed by MR thermometry (MRT). A fiber optic temperature monitoring device was used to measure the temperature in real-time during the entire heating period with online adjustment of heat delivery by alternating the laser power. Drug delivery was optimized under magnetic resonance (MR) image guidance by co-encapsulation of an MR contrast agent (i.e.,&#160;gadoteridol) along with CDDP into the thermosensitive liposomes as a means to validate the heating protocol and to assess tumor accumulation. The heating protocol consisted of a preheating period of 5 min prior to administration of HTLC and 20 min heating post-injection. This heating protocol resulted in effective release of the encapsulated agents with the highest MR signal change observed in the heated tumor in comparison to the unheated tumor and muscle. This study demonstrated the successful application of the laser-based heating apparatus for preclinical thermosensitive liposome development and the importance of MR-guided validation of the heating protocol for optimization of drug delivery.

AN MRI-compatible&#160;custom-designed laser-based heating apparatus has been developed to provide local heating of subcutaneous tumors in order to activate release of agents from thermosensitive liposomes specifically at the tumor region.

Appositamente studiata apparecchi di riscaldamento a base laser per la Release Innescata di Cisplatino da termosensibile liposomi con Risonanza Magnetica Guidance Immagine

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect ...

Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for specialized equipment and trained personnel require that many diagnostic tests be performed at remote, centralized clinical laboratories. Magnetic levitation is a simple yet powerful technique and can be applied to levitate cells, which are suspended in a paramagnetic solution and placed in a magnetic field, at a position determined by equilibrium between a magnetic force and a buoyancy force. Here, we present a versatile platform technology designed for point-of-care diagnostics which uses magnetic levitation coupled to microscopic imaging and automated analysis to determine the density distribution of a patient's cells as a useful diagnostic indicator. We present two platforms operating on this principle: (i) a smartphone-compatible version of the technology, where the built-in smartphone camera is used to image cells in the magnetic field and a smartphone application processes the images and to measures the density distribution of the cells and (ii) a self-contained version where a camera board is used to capture images and an embedded processing unit with attached thin-film-transistor (TFT) screen measures and displays the results. Demonstrated applications include: (i) measuring the altered distribution of a cell population with a disease phenotype compared to a healthy phenotype, which is applied to sickle cell disease diagnosis, and (ii) separation of different cell types based on their characteristic densities, which is applied to separate white blood cells from red blood cells for white blood cell cytometry. These applications, as well as future extensions of the essential density-based measurements enabled by this portable, user-friendly platform technology, will significantly enhance disease diagnostic capabilities at the point of care.

We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

Levitazione magnetica Accoppiato con Imaging portatile e analisi per la malattia di diagnostica

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

<meta charset="utf8"></head><body>Saggio fluorescente per la rilevazione di nuovi chelanti del ferro

Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target for anti-cancer approaches. The development of novel compounds and the identification of leads for further modification requires that proof of mechanism assays be carried out. There are many assays to evaluate the impact on proliferation; however, the ability to chelate iron is an important and sometimes overlooked end-point measure due to the high costs of equipment and the challenge to quickly and reproducibly quantify the strength of chelation. Here, we describe a quantifiable and inexpensive cell-free fluorescent method to confirm the ability of novel compounds to chelate iron. Our assay relies on the commercially available inexpensive fluorescent dye Calcein, whose fluorescence can be quantified on most fluorescent microtiter plate readers. Calcein is a weak iron chelator, and its fluorescence is quenched when it binds Fe2+/3+; fluorescence is restored when a novel chelator outcompetes Calcein for bound Fe2+/3+. The removal of fluorescent quenching and the resulting increase in fluorescence allows the chelation ability of a novel putative chelator to be determined. Therefore, we offer an inexpensive, high-throughput assay that allows the rapid screening of novel candidate chelator compounds.

<meta charset="utf8"></head><body>Autore in primo piano: Valutazione dell'impatto di nuovi chelanti del ferro sul metabolismo delle cellule tumorali

We describe a fluorescent assay that can quickly and inexpensively confirm the ability of novel compounds to chelate iron. The assay measures the ability of compounds to outcompete the iron binding activity of the weak iron chelating fluorescent probe Calcein, resulting in a quantifiable increase in fluorescence when chelation occurs.

Metodo fluorescente quantificabile ed economico per confermare la capacità di nuovi composti di chelare il ferro 

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target ...

Ricerca sul cancro

Automated Preparation of [68Ga]Ga-3BP-3940 on a Synthesis Module for PET Imaging of the Tumor Microenvironment

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe targeting the fibroblast activation protein for positron emission tomography imaging of the tumor microenvironment. The reaction conditions involved acetate buffer (final concentration: 0.1 M), methionine as an anti-radiolysis agent (final concentration: 5.4 mg/mL), and 30 &#181;g of 3BP-3940, with heating for 8 min at 98 &#176;C. A final purification step on a C18 cartridge was necessary to obtain a radiolabeled product of high purity. In contrast, the generator-produced 68Ga was used directly without a concentration step on a cation exchange cartridge. The production of three validation batches confirmed the method&#39;s reliability, allowing the synthesis of [68Ga]Ga-3BP-3940 in 22.3 &#177; 0.6 min with high radiochemical purity (RCP), as determined by both radio-HPLC (99.1% &#177; 0.1%) and radio-TLC (99.2% &#177; 0.1%). The average radiochemical yield, based on RCP values measured by radio-HPLC, was 74.4% &#177; 3.3%. The stability of the radiolabeled product was demonstrated for up to 4 h after preparation. This protocol provides a reliable, rapid, and efficient methodology for the preparation of [68Ga]Ga-3BP-3940, which can easily be transposed to a clinical setting.

This research describes the automated process for [68Ga]Ga-3BP-3940 production with the GAIA V2 synthesizer, for PET imaging of fibroblast activation protein. The results of quality control tests performed on three test batches are also presented.

Preparazione automatizzata di [68Ga]Ga-3BP-3940 su un modulo di sintesi per l'imaging PET del microambiente tumorale

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe ...

Un sensore Aptamero-based per Unchelated gadolinio (III)

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Indagini su GA (III) Complesso di EOB-DTPA e la sua

Preparazione e

MR Imaging Molecolare del cancro alla prostata con un piccolo Molecular CLT1 Peptide mirato di contrasto agente

Biofunctionalized Prussian Blue Nanoparticelle per applicazioni di imaging multimodale molecolari

Appositamente studiata apparecchi di riscaldamento a base laser per la Release Innescata di Cisplatino da termosensibile liposomi con Risonanza Magnetica Guidance Immagine

Levitazione magnetica Accoppiato con Imaging portatile e analisi per la malattia di diagnostica

Metodo fluorescente quantificabile ed economico per confermare la capacità di nuovi composti di chelare il ferro

Metodo fluorescente quantificabile ed economico per confermare la capacità di nuovi composti di chelare il ferro

Metodo fluorescente quantificabile ed economico per confermare la capacità di nuovi composti di chelare il ferro

Preparazione automatizzata di [⁶⁸Ga]Ga-3BP-3940 su un modulo di sintesi per l'imaging PET del microambiente tumorale