combining-qd-fret-microfluidics-to-monitor-dna-nanocomplex-self

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

In this study, the authors tested the feasibility of using ultrasonography (US) to monitor catheter-based vascular gene microsphere delivery. Polymeric biodegradable microspheres (mean diameter, 5 microm) were prepared by using a double-emulsion technique to encapsulate DNA-plasmid-encoding green fluorescent protein (GFP) genes. With use of gene-delivery catheters, GFP microspheres were locally delivered into the left femoral arterial walls of six pigs; the contralateral arteries were not infused with microspheres and thus served as negative control vessels. The delivery procedures were monitored with high-frequency (8-15-MHz) transducer US. The effectiveness of monitoring with US was compared with the effectiveness of monitoring with immunohistochemical anti-GFP staining. A highly echogenic "star burst" sign around the entire vessel wall was seen at US and correlated with immunohistochemical findings that showed the destination of the gene microspheres. Study results demonstrate the potential of US for monitoring catheter-based vascular gene microsphere delivery in vivo.

In vivo US monitoring of catheter-based vascular delivery of gene microspheres in pigs: feasibility.

In-vivo-&#xDC;berwachung von US-Katheter-basierte vaskul&#xE4;re Abgabe von Gen-Mikrosph&#xE4;ren in Schweinen: Machbarkeit.

We evaluate the in vivo use of an optical imaging method to detect the vascular expression of green fluorescent protein (GFP) or red fluorescent protein (RFP), and to detect the simultaneous expression of GFP and RFP after transduction into arteries by a dual-promoter lentiviral vector driving their concurrent expression. This method involves using a charge-coupled device camera to detect fluorescence, a fiber optic probe to transmit light, and optical filters to distinguish each marker. In animal models, these vectors are locally delivered to target arteries, whereas the gene for a nonfluorescent cell-surface protein is transduced into contralateral arteries as the sham control. The images show distinct areas of bright fluorescence from GFP and RFP along the target arteries on excitation; no exogenous fluorescence is observed in the controls. Measured signal intensities from arteries transduced with the single- and dual-promoter vectors exceed the autofluorescence signal from the controls. Transgene expression of GFP and RFP in vivo is confirmed with confocal microscopy. We demonstrate the use of an optical imaging method to concurrently detect two distinct fluorescent proteins, potentially permitting the expression of multiple transgenes and their localization in the vasculature to be monitored.

Detection of dual-gene expression in arteries using an optical imaging method.

Nachweis von Dual-Genexpression in Arterien unter Verwendung eines optischen Bildgebungsverfahren.

Gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) was encapsulated into biodegradable, bioadhesive polymeric microparticles to enable noninvasive monitoring of their local intravesical delivery with MRI. The microparticles were characterized by contrast agent encapsulation and release kinetics, T(1) relaxation rates, and contrast enhancement in vivo. The level of Gd-DTPA loading into microparticles was 14.3 +/- 0.6 mug/mg polymer. The measured T(1) relaxation rates of the microparticles showed a direct dependence on Gd-DPTA content. Both 1.5T and 4.7T MR scanners were used to image murine bladders instilled intravesically with Gd-DTPA-loaded particles in vivo. MR images showed ring-shaped regions of enhancement inscribing the bladder wall, which were attributed to the microparticles that were preferentially adherent to the mucosa lining the urothelium. The images of controls exhibited no such enhancement. The normalized signal intensities measured from post-instillation images were significantly greater (P < 0.05) than those in the pre-instillation images. Contrast enhancement was observed for at least 5 days after the initial instillation, although the enhancement decreased due to microparticle degradation or mucosa renewal. The localized distribution of biodegradable, bioadhesive microparticles encapsulating Gd-DTPA was successfully visualized with MRI in vivo, allowing particle-mediated delivery to be temporally and spatially monitored noninvasively.

MR imaging of biodegradable polymeric microparticles: a potential method of monitoring local drug delivery.

MR-Bildgebung von biologisch abbaubaren polymeren Mikropartikeln: eine potentielle Verfahren zur &#xDC;berwachung local drug delivery.

Recent results from animal studies suggest that stem cells may be able to home to sites of myocardial injury to assist in tissue regeneration. However, the histological interpretation of postmortem tissue, on which many of these studies are based, has recently been widely debated.

Dynamic imaging of allogeneic mesenchymal stem cells trafficking to myocardial infarction.

Dynamische Bildgebung von allogenen mesenchymalen Stammzellen Handel von Myokardinfarkt.

We demonstrate a highly sensitive method to characterize the structural composition and intracellular fate of polymeric DNA nanocomplexes, formed by condensing plasmid DNA with cationic polymers through electrostatic interactions. Rational design of more efficient polymeric gene carriers will be possible only with mechanistic insights of the rate-limiting steps in the non-viral gene transfer process. To characterize the composition and binding dynamics of nanocomplexes, plasmid and its polymer carrier within nanocomplexes were labeled with quantum dots (QDs) and fluorescent organic dyes, respectively, as a donor and acceptor pair for fluorescence resonance energy transfer (FRET). The high signal-to-noise ratio in QD-mediated FRET enabled precise detection of discrete changes in nanocomplex state at the single-particle level, against various intracellular microenvironments. The distribution and unpacking of individual nanocomplexes within cells could thus be unambiguously followed by fluorescence microscopy. QD-FRET is a highly sensitive and quantitative method to determine the composition and dynamic stability of nanocomplexes during intracellular transport, where barriers to gene delivery may be identified to facilitate gene carrier optimization.

Evaluating the intracellular stability and unpacking of DNA nanocomplexes by quantum dots-FRET.

Wir zeigen eine hochempfindliche Methode, um die strukturelle Zusammensetzung und intrazellul&auml;re Schicksal von Polymeren DNA Nanokomplexe, gebildet durch Kondensation von Plasmid-DNA mit kationischen Polymeren durch elektrostatische Wechselwirkungen charakterisieren. Rationales Design effizienter Polymeren gen-Tr&auml;ger ist nur mit mechanistische Einblicke der geschwindigkeitsbestimmende Schritte in der nicht-virale Gentransfer-&Uuml;bertragung m&ouml;glich. Um die Zusammensetzung und die Bindung Dynamik der Nanokomplexe zu charakterisieren, wurden Plasmid und seine Polymer Frachtf&uuml;hrer Nanokomplexe mit Quantenpunkte (QDs) und fluoreszierende organische Farbstoffe, bzw. als Donor und Akzeptor-Paar f&uuml;r Fluoreszenz-Resonanz-Energie-Transfer (FRET) bezeichnet. Das hohe Signal-Rausch-Verh&auml;ltnis in QD-vermittelte FRET aktiviert pr&auml;zise Erkennung von diskreten &Auml;nderungen in Nanokomplexe Zustand auf Einzel-Partikel-Ebene gegen verschiedene intrazellul&auml;re Mikroumgebungen. Die Verteilung und Auspacken von einzelnen Nanokomplexe innerhalb von Zellen k&ouml;nnten somit eindeutig Fluoreszenzmikroskopie folgen. QD-Bund ist eine hochempfindliche und quantitative Methode zur Bestimmung der Zusammensetzung und dynamische Stabilit&auml;t des Nanokomplexe beim intrazellul&auml;ren Transport, wo k&ouml;nnen Handelshemmnisse gen Lieferung festgestellt werden, Gen-Tr&auml;ger-Optimierung zu erleichtern.

Bewertung der intrazellul&auml;ren Stabilit&auml;t und Auspacken von DNA Nanokomplexe von Quantum Dots-FRET.

Quantum-dots-FRET nanosensors for detecting unamplified nucleic acids by single molecule detection.

Quanten-Punkte-FRET Nanosensoren und zum Erkennen von unverst&auml;rkt Nukleins&auml;uren von Einzelmolek&uuml;l-Erkennung.

A major challenge for non-viral gene delivery is gaining a mechanistic understanding of the rate-limiting steps. A critical barrier in polyplex-mediated gene delivery is the timely unpacking of polyplexes within the target cell to liberate DNA for efficient gene transfer. In this study, the component plasmid DNA and polymeric gene carrier were individually labeled with quantum dots (QDs) and Cy5 dyes, respectively, as a donor and acceptor pair for fluorescence resonance energy transfer (FRET). The high signal-to-noise ratio in QD-mediated FRET enabled sensitive detection of discrete changes in polyplex stability. The intracellular uptake and dissociation of polyplexes through QD-FRET was captured over time by confocal microscopy. From quantitative image-based analysis, distributions of released plasmid within the endo/lysosomal, cytosolic, and nuclear compartments formed the basis for constructing a three-compartment first-order kinetics model. Polyplex unpacking kinetics for chitosan, polyethylenimine, and polyphosphoramidate were compared and found to correlate well with transfection efficiencies. Thus, QD-FRET-enabled detection of polyplex stability combined with image-based quantification is a valuable method for studying mechanisms involved in polyplex unpacking and trafficking within live cells. We anticipate that this method will also aid the design of more efficient gene carriers.

Quantitative comparison of intracellular unpacking kinetics of polyplexes by a model constructed from quantum dot-FRET.

Eine gro&szlig;e Herausforderung f&uuml;r nicht-virale Gentransfer-Lieferung gewinnt eine mechanistische Verst&auml;ndnis der geschwindigkeitsbestimmende Schritte. Eine wichtige Barriere Polyplex-vermittelte gen Lieferung ist die rechtzeitige Auspacken von Polyplexes innerhalb der Zielzelle DNA f&uuml;r effiziente Gentransfer zu befreien. In dieser Studie wurden die Komponente Plasmid-DNA und Polymeren gen Tr&auml;ger individuell mit Quantenpunkte (QDs) und Cy5 Farbstoffe, bzw. als Donor und Akzeptor-Paar f&uuml;r Fluoreszenz-Resonanz-Energie-Transfer (FRET) bezeichnet. Das hohe Signal-Rausch-Verh&auml;ltnis in QD-vermittelte FRET aktiviert empfindlichen Nachweis von diskreten &Auml;nderungen in Polyplex Stabilit&auml;t. Die intrazellul&auml;re Aufnahme und Dissoziation von Polyplexes durch QD-FRET geriet im Laufe der Zeit confocal Mikroskopie. Aus quantitativen bildbasierte Analyse gebildet Verteilungen von ver&ouml;ffentlichten Plasmid innerhalb der Endo/lysosomale, Zytosolische, nukleare F&auml;cher und die Grundlage f&uuml;r den Bau eines drei-Fach erster Ordnung Kinetik-Modells. Polyplex Auspacken Kinetik f&uuml;r Chitosan, wurden Polyethylenimin und Polyphosphoramidate verglichen und korrelieren gut mit Transfektion Effizienz gefunden. QD-FRET-f&auml;higen Nachweisvon Polyplex Stabilit&auml;t kombiniert mit Image-basierte Quantifizierung deshalb eine wertvolle Methode f&uuml;r einstudieren Mechanismen Polyplex Auspacken und Menschenhandel in lebenden Zellen beteiligt. Wir erwarten, dass diese Methode auch die Gestaltung effizienter gen Tr&auml;ger helfen werden.

Quantitativer Vergleich der intrazellul&auml;ren Auspacken Kinetik der Polyplexes durch ein Modell gebaut von Quantum Dot-FRET.

We present a novel convergence of quantum-dot-mediated fluorescence resonance energy transfer (QD-FRET) and microfluidics, through which molecular interactions were precisely controlled and monitored using highly sensitive quantum-dot-mediated FRET. We demonstrate its potential in studying the kinetics of self-assembly of DNA polyplexes under laminar flow in real time with millisecond resolution. The integration of nanophotonics and microfluidics offers a powerful tool for elucidating the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

The convergence of quantum-dot-mediated fluorescence resonance energy transfer and microfluidics for monitoring DNA polyplex self-assembly in real time.

Pr&auml;sentieren wir Ihnen eine neuartige Konvergenz der Quantum-Dot-vermittelter Fluoreszenz-Resonanz-Energie-Transfer (QD-FRET) und Microfluidics, durch welche molekularen Wechselwirkungen waren genau gesteuert und &uuml;berwacht mit hochsensiblen Quantum-Dot-vermittelte FRET. Wir zeigen ihr Potential in die Kinetik der Selbstorganisation von DNA Polyplexes unter Laminar-Flow in Echtzeit mit einer Millisekunde Aufl&ouml;sung zu studieren. Die Integration der Nanophotonik und Mikrofluidik bietet ein leistungsf&auml;higes Werkzeug f&uuml;r die elucidating der Bildung von Polyelectrolyte Polyplexes, die erwartet wird, um bessere Kontrolle und Synthese von einheitlichen und anpassbare Polyplexes f&uuml;r zuk&uuml;nftige Nukleins&auml;ure-basierten Therapeutika bereitzustellen.

Die Konvergenz der Quantum-Dot-vermittelter Fluoreszenz-Resonanz-Energie-Transfer und Biochips zur &Uuml;berwachung der DNA Polyplex Selbstmontage in Echtzeit.

Nanoscale vectors comprised of cationic polymers that condense DNA to form nanocomplexes are promising options for gene transfer. The rational design of more efficient nonviral gene carriers will be possible only with better mechanistic understanding of the critical rate-limiting steps, such as nanocomplex unpacking to release DNA and degradation by nucleases. We present a two-step quantum dot fluorescence resonance energy transfer (two-step QD-FRET) approach to simultaneously and non-invasively analyze DNA condensation and stability. Plasmid DNA, double-labeled with QD (525 nm emission) and nucleic acid dyes, were complexed with Cy5-labeled cationic gene carriers. The QD donor drives energy transfer stepwise through the intermediate nucleic acid dye to the final acceptor Cy5. At least three distinct states of DNA condensation and integrity were distinguished in single particle manner and within cells by quantitative ratiometric analysis of energy transfer efficiencies. This novel two-step QD-FRET method allows for more detailed assessment of the onset of DNA release and degradation simultaneously.

Simultaneous Non-invasive Analysis of DNA Condensation and Stability by Two-step QD-FRET.

Nanoskalige Vektoren bestehend aus kationische Polymere, die DNA in Form Nanokomplexe kondensieren sind vielversprechende Optionen Gentransfer. Das rationale Design von effizienter nonviral gen-Tr&auml;gern ist nur mit besser mechanistische Verst&auml;ndnis der kritischen geschwindigkeitsbestimmende Schritte, wie Nanokomplexe Auspacken um DNA und Abbau von Nukleasen Version m&ouml;glich. Wir pr&auml;sentieren einen zweistufigen Quantum Dot Fluoreszenz Resonanz Energie Transfer (zweistufiger QD-FRET) Ansatz gleichzeitig und nicht-invasiv Analyse DNA-Kondensation und Stabilit&auml;t. Plasmid-DNA, Doppel-beschriftet mit QD (525 nm Emission) und Nukleins&auml;ure-Farbstoffe, komplexiert mit kationischen gen Cy5 beschriftet wurden. Der QD-Spender Laufwerke Energietransfer schrittweise durch die Mittelstufe Nukleins&auml;ure-Farbstoff auf der endg&uuml;ltigen Akzeptor Cy5. Mindestens drei verschiedene Staaten von DNA-Kondensation und Integrit&auml;t wurden in einzelne Teilchen Weise und innerhalb der Zellen durch quantitative ratiometrisch Analyse der &Uuml;bertragung Energieeffizienz ausgezeichnet. Diese neuartigen zweistufigen QD-FRET-Methode erm&ouml;glicht gleichzeitig eine eingehendere Beurteilung des Eintritts der DNA-Release und Abbau.

Article	Year
In vivo US monitoring of catheter-based vascular delivery of gene microspheres in pigs: feasibility. Radiology\| PubMed ID: 12893906	2003
Detection of dual-gene expression in arteries using an optical imaging method. Journal of biomedical optics\| PubMed ID: 15568943	2004
MR imaging of biodegradable polymeric microparticles: a potential method of monitoring local drug delivery. Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine / Society of Magnetic Resonance in Medicine\| PubMed ID: 15723408	2005
Dynamic imaging of allogeneic mesenchymal stem cells trafficking to myocardial infarction. Circulation\| PubMed ID: 16129797	2005
Evaluating the intracellular stability and unpacking of DNA nanocomplexes by quantum dots-FRET. Journal of controlled release : official journal of the Controlled Release Society\| PubMed ID: 17081642	2006
Quantum-dots-FRET nanosensors for detecting unamplified nucleic acids by single molecule detection. Nanomedicine (London, England)\| PubMed ID: 17716215	2006
Quantitative comparison of intracellular unpacking kinetics of polyplexes by a model constructed from quantum dot-FRET. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 18180773	2008
The convergence of quantum-dot-mediated fluorescence resonance energy transfer and microfluidics for monitoring DNA polyplex self-assembly in real time. Nanotechnology\| PubMed ID: 19417478	2009
Simultaneous Non-invasive Analysis of DNA Condensation and Stability by Two-step QD-FRET. Nano today\| PubMed ID: 20161048	2009
Dual-sensitive micellar nanoparticles regulate DNA unpacking and enhance gene-delivery efficiency. Advanced materials (Deerfield Beach, Fla.)\| PubMed ID: 20440698	2010

Hunter H. Chen

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