Hunter H. Chen

Quantitative Comparison of Intracellular Unpacking Kinetics of Polyplexes by a Model Constructed From Quantum Dot-FRET

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.

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

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, T1 relaxation rates, and contrast enhancement in vivo. The level of Gd‐DTPA loading into microparticles was 14.3 ± 0.6 μg/mg polymer. The measured T1 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. Magn Reson Med 53:614–620, 2005.

Dual-sensitive micellar nanoparticles regulate DNA unpacking and enhance gene-delivery efficiency.

Polymer-based gene carriers have been increasingly proposed as a safer alternative to viral vectors, due to their ease of synthesis, flexibility in the size of the transgene to be delivered, and minimal host immune responses. A major challenge to apply polycation/DNA nanoparticles in vivo, however, is the poor colloidal stability and serum stability, which lead to rapid aggregation followed by macrophage uptake, and premature dissociation of nanoparticles and release of DNA payload, respectively. Together they conspire to yield extremely low gene delivery efficiency through intravenous injection. To improve efficiency, an ideal polycation/DNA delivery system should satisfy the conflicting requirements of high stability in extracellular environment and endolysosomal compartments, so that the nanoparticles maintain their small size and integrity in circulation and endosomal sequestration, followed by low stability in cytosol and nucleus so as to allow for DNA release and transcription.

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

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.

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

Evaluation of: Zhang CY, Yeh HC, Kuroki MT, Wang TH: Single-quantum-dot-based DNA nanosensor. Nat. Mater. 4(11), 826–831 (2005) [1]. Quantitative and sensitive detection of minute copies of nucleic acid sequences is critical in diagnosing disease and in understanding biomolecular processes. An inorganic–organic hybrid fluorescence resonance energy transfer (FRET) nanosensor based on quantum dots (QDs) was developed to overcome the limitations of conventional FRET-based probes. Functionalized QDs served as FRET donors and as nanoassemblies that can couple to multiple targets hybridized as a sandwich between a capture probe and a reporter probe. Target sequences are detected directly in solution by single molecule detection (SMD) without prior separation or amplification. This system is sensitive enough to detect approximately 50 or fewer copies and to discriminate point mutations. QD-FRET and SMD are platform technologies that will find many applications for detecting biomarkers or studying various biomole...

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

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.

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 functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships.

Development of a non-invasive optical imaging method for tracking vascular gene expression

Gene therapy is an exciting frontier in modern medicine. To date, no imaging modalities are available for monitoring vascular gene therapy. Green fluorescent protein (GFP) has become an increasingly common marker for gene therapy. We have developed an optical imaging method to track vascular gene expression by detecting fluorescence emitted from GFP or red fluorescent protein (RFP) in arterial walls following gene transfer. We surgically transferred GFP- and RFP-vectors into the femoral and carotid arteries of three New Zealand white rabbits. Excitation light was transmitted through a fiber-optic ring-light (Nevoscope) and GFP and RFP fluorescence was detected by a charge coupled device (CCD) camera. Direct contact images of the target arteries demonstrated that this method was capable of both discriminating between normal and transferred arterial tissues and mapping fluorescent protein localization. Subsequent measurements by confocal microscopy showed statistically significant differences in average fluorescent signal intensity between the control and transferred tissues. This result was corroborated by immunohistochemical staining. These preliminary results are encouraging evidence that the optical imaging,method can be developed further to be performed non-invasively and in vivo in a clinical setting.

Quantitative kinetic analysis of DNA nanocomplex self-assembly with Quantum Dots FRET in a microfluidic device

The demand for safer and more efficient non-viral gene vectors has increased with the recent progress of genetic medicine. Appropriate nanocomplex assembly of DNA and gene carriers is critical for successful cellular entry and transfection. However, there is a lack of knowledge on this self-assembly process, let alone the controllability of monodisperse nanocomplexes. This paper describes a novel platform integrating nanobiophotonics (quantum dots-mediated FRET) and microfluidic technology to determine binding kinetics that govern the structural and chemical properties of DNA nanocomplexes. We anticipate that this method will elucidate mechanistic and kinetic insights into the self-assembly process of nanocomplexes which may facilitate the rational design of more efficient gene carriers. In addition, a microfluidic platform offers many advantages, including small volume, fast response to external stimulations, continuous monitoring and real-time control of reaction environments, which may be potentially used to generate more monodisperse complexes.

Optical imaging of green fluorescent protein markers for tracking vascular gene expression: a feasibility study in human tissue-like phantoms

Vascular gene therapy is an exciting approach to the treatment of cardiovascular diseases. However, to date, there are no imaging modalities available for non-invasive detection of vascular gene expression. We have developed an optical imaging method to track vascular gene expression by detecting fluorescent signals emitted from arterial walls following gene transfer. To investigate the feasibility of this new technique, we performed experiments on a set of human tissue-like phantoms using a common biological marker in gene therapy, the green fluorescent protein (GFP). The phantoms were constructed to mimic the arterial geometry beneath a tissue layer. Human smooth muscle cells transfected with GFP were embedded in a capillary tube in the phantom. Monte Carlo modeling of the phantom experiment was performed to optimize the performance of the optical imaging system. We compared the fluence rates among three types of light beams, including ring beam, Gaussian beam, and flat beam. The results showed that our optical imaging system was able to detect fluorescent signals up to 5-mm depth in the phantom, and that flat beam geometry would produce the optimum fluorescence remittance. This study provides valuable insights for improvements to the optical imaging system and refinement of the new technique to non-invasively detect/track vascular gene expression.