Tristan Tabouillot

Encapsulation of Organic Molecules in Calcium Phosphate Nanocomposite Particles for Intracellular Imaging and Drug Delivery

Encapsulation of imaging agents and drugs in calcium phosphate nanoparticles (CPNPs) has potential as a nontoxic, bioresorbable vehicle for drug delivery to cells and tumors. The objectives of this study were to develop a calcium phosphate nanoparticle encapsulation system for organic dyes and therapeutic drugs so that advanced fluoresence methods could be used to assess the efficiency of drug delivery and possible mechanisms of nanoparticle bioabsorption. Highly concentrated CPNPs encapsulating a variety of organic fluorophores were successfully synthesized. Well-dispersed CPNPs encapsulating Cy3 amidite exhibited nearly a 5-fold increase in fluorescence quantum yield when compared to the free dye in PBS. FCS diffusion data and cell staining were used to show pH-dependent dissolution of the particles and cellular uptake, respectively. Furthermore, an experimental hydrophobic cell growth inhibitor, ceramide, was successfully delivered in vitro to human vascular smooth muscle cells via encapsulation in CPNPs. These studies demonstrate that CPNPs are effective carriers of dyes and drugs for bioimaging and, potentially, for therapeutic intervention.

Enzyme Molecules as Nanomotors

Using fluorescence correlation spectroscopy, we show that the diffusive movements of catalase enzyme molecules increase in the presence of the substrate, hydrogen peroxide, in a concentration-dependent manner. Employing a microfluidic device to generate a substrate concentration gradient, we show that both catalase and urease enzyme molecules spread toward areas of higher substrate concentration, a form of chemotaxis at the molecular scale. Using glucose oxidase and glucose to generate a hydrogen peroxide gradient, we induce the migration of catalase toward glucose oxidase, thereby showing that chemically interconnected enzymes can be drawn together.

Integrated multimodal microscopy, time-resolved fluorescence, and optical-trap rheometry: toward single molecule mechanobiology

Cells respond to forces through coordinated biochemical signaling cascades that originate from changes in single-molecule structure and dynamics and proceed to large-scale changes in cellular morphology and protein expression. To enable experiments that determine the molecular basis of mechanotransduction over these large time and length scales, we construct a confocal molecular dynamics microscope (CMDM). This system integrates total-internal-reflection fluorescence (TIRF), epifluorescence, differential interference contrast (DIC), and 3-D deconvolution imaging modalities with time-correlated single-photon counting (TCSPC) instrumentation and an optical trap. Some of the structures hypothesized to be involved in mechanotransduction are the glycocalyx, plasma membrane, actin cytoskeleton, focal adhesions, and cell-cell junctions. Through analysis of fluorescence fluctuations, single-molecule spectroscopic measurements [e.g., fluorescence correlation spectroscopy (FCS) and time-resolved fluorescence] can be correlated with these subcellular structures in adherent endothelial cells subjected to well-defined forces. We describe the construction of our multimodal microscope in detail and the calibrations necessary to define molecular dynamics in cell and model membranes. Finally, we discuss the potential applications of the system and its implications for the field of mechanotransduction.

Monitoring cellular mechanosensing using time-correlated single photon counting

Endothelial cells (ECs) convert mechanical stimuli into chemical signaling pathways to regulate their functions and properties. It is hypothesized that perturbation of cellular structures by force is accompanied by changes in molecular dynamics. In order to address these fundamental issues in mechanosensation and transduction, we have developed a hybrid multimodal microscopy - time-correlated single photon counting (TCSPC) spectroscopy system intended to determine time- and position dependent mechanically-induced changes in the dynamics of molecules in live cells as determined from fluorescence lifetimes and autocorrelation analysis (fluorescence correlation spectroscopy). Colocalization of cell-structures and mechanically-induced changes in molecular dynamics can be done in post-processing by comparing TCSPC data with 3-D models generated from total internal reflection fluorescence (TIRF), differential interference contrast (DIC), epifluorescence, and deconvolution. We present control experiments in which the precise location of the apical cell membrane with respect to a confocal probe is assessed using information obtainable only from TCSPC. Such positional accuracy of TCSPC measurements is essential to understanding the role of the membrane in mechanotransduction. We predict that TCSPC will become a useful method to obtain high temporal and spatial resolution information on localized mechanical phenomena in living endothelial cells. Such insight into mechanotransduction phenomenon may uncover the origins of mechanically-related diseases such as atherosclerosis.

Time-Correlated, Single-Photon Counting Methods in Endothelial Cell Mechanobiology

While mechanical forces are known to guide the development of nearly all biological tissues including bone, cartilage, and many soft tissues, much attention has focused on endothelial cell mechanobiology and the role of blood flow-induced forces in regulating the health of blood vessels. It is now well accepted that modulation of endothelial cell physiology and pathophysiology by fluid mechanical forces is a principal reason why atherosclerotic lesions are located at areas of disturbed flow including at arterial branch points and areas of high arterial curvature. However, the molecular identity of endothelial cell mechanosensors remains elusive largely due to the complexity of cell mechanics and to the difficulty in identifying when and where a candidate mechanosensor has been perturbed. Thus, new methods of cell-specific mechanical modeling along with molecular-scale readouts of perturbation by force are needed to help unravel the magnitude-, time-, and position-dependent responses of endothelial cells to mechanical forces.

Photophysical characterization of Dye-Encapsulated Calcium Phosphate Nanoparticles

Organic dyes exhibit rapid photobleaching, low quantum yield, and random blinking under physiological conditions, limiting their utility in in vivo imaging. To address these photophysical shortcomings, the Adair group at Penn State has recently developed a novel method for synthesizing dye-encapsulated calcium phosphate (CP) nanoparticles based on a double microemulsion method. In this study, time-resolved single photon counting methods were used to characterize cy3-encapsulted CP nanoparticle size, dispersity, molecular brightness, and fluorescence lifetime (FL). Particle sizes measured using fluorescence correlation spectroscopy (FCS) confirmed the presence of highly mono-disperse 20 nm particles. The brightness of an individual nanoparticle measured using moment analysis was found to be 20 times higher than the free dye, due to a five-fold increase in quantum efficiency and encapsulation of 4 dye molecules per particle. FL of the encapsulated dye was independent of the solvent (water, PBS, DMSO, and 50% glycerol), suggesting that the dye was well-protected in the CP matrix. Furthermore, increased FL in CP nanoparticles compared to free dye suggests that the photoisomerization of cy3 was inhibited due to restricted mobility of the dye in CP matrix. Photostability increased 50-fold likely because the dye was protected from the photobleaching effects of dissolved oxygen. Finally, systemic administration of PEGylated CP nanoparticles in nude mice implanted with breast cancer tumors retained fluorescence signal in tumors even after 96 hours post-injection, demonstrating the utility of CP nanoparticles for long term in vivo imaging.

SHEAR STRESS INDUCES TIME- AND DOMAIN- DEPENDENT CHANGES IN LIPID DYNAMICS OF ENDOTHELIAL CELL MEMBRANES

Endothelial cells (ECs) form the inner lining of the blood vasculature and are exposed to shear stress (τ), the tangential component of hemodynamic forces. ECs transduce τ into biochemical signals possibly via EC-membrane perturbations. We have previously used confocal-FRAP on the DiI-stained plasma membranes of confluent cultured bovine aortic ECs (BAECs) to show that τ induces a rapid, spatially heterogeneous, and time-dependent increase in the lateral diffusion of the fluorescent lipoid probe in the BAEC membrane [1]. We now present evidence at the single molecule level that shear stress differentially perturbs membrane domains that are defined by their selective staining by lipoid dyes (DiI) of differing alkyl chain lengths. This study is the first to directly measure perturbation by shear stress of endothelial cell membrane microdomains.Copyright