Portable bioluminescent platform for in vivo monitoring of biological processes in non-transgenic animals.

Bioluminescent imaging (BLI) is one of the most powerful and widely used preclinical imaging modalities. However, the current technology relies on the use of transgenic luciferase-expressing cells and animals and therefore can only be applied to a limited number of existing animal models of human disease. Here, we report the development of a "portable bioluminescent" (PBL) technology that overcomes most of the major limitations of traditional BLI. We demonstrate that the PBL method is capable of noninvasive measuring the activity of both extracellular (e.g., dipeptidyl peptidase 4) and intracellular (e.g., cytochrome P450) enzymes in vivo in non-luciferase-expressing mice. Moreover, we successfully utilize PBL technology in dogs and human cadaver, paving the way for the translation of functional BLI to the noninvasive quantification of biological processes in large animals. The PBL methodology can be easily adapted for the noninvasive monitoring of a plethora of diseases across multiple species.

A bioluminescent probe for longitudinal monitoring of mitochondrial membrane potential.

Mitochondrial membrane potential (ΔΨm) is a universal selective indicator of mitochondrial function and is known to play a central role in many human pathologies, such as diabetes mellitus, cancer and Alzheimer's and Parkinson's diseases. Here, we report the design, synthesis and several applications of mitochondria-activatable luciferin (MAL), a bioluminescent probe sensitive to ΔΨm, and partially to plasma membrane potential (ΔΨp), for non-invasive, longitudinal monitoring of ΔΨm in vitro and in vivo. We applied this new technology to evaluate the aging-related change of ΔΨm in mice and showed that nicotinamide riboside (NR) reverts aging-related mitochondrial depolarization, revealing another important aspect of the mechanism of action of this potent biomolecule. In addition, we demonstrated application of the MAL probe for studies of brown adipose tissue (BAT) activation and non-invasive in vivo assessment of ΔΨm in animal cancer models, opening exciting opportunities for understanding the underlying mechanisms and for discovery of effective treatments for many human pathologies.

Preclinical Evaluation of Photoacoustic Imaging as a Novel Noninvasive Approach to Detect an Orthopaedic Implant Infection.

INTRODUCTION: Diagnosing prosthetic joint infection (PJI) poses significant challenges, and current modalities are fraught with low sensitivity and/or potential morbidity. Photoacoustic imaging (PAI) is a novel ultrasound-based modality with potential for diagnosing PJI safely and noninvasively. MATERIALS: In an established preclinical mouse model of bioluminescent Staphylococcus aureus PJI, fluorescent indocyanine green (ICG) was conjugated to ß-cyclodextrin (CDX-ICG) or teicoplanin (Teic-ICG) and injected intravenously for 1 week postoperatively. Daily fluorescent imaging and PAI were used to localize and quantify tracer signals. Results were analyzed using 2-way analysis of variance. RESULTS: Fluorescence clearly localized to the site of infection and was significantly higher with Teic-ICG compared with CDX-ICG (P = 0.046) and ICG alone (P = 0.0087). With PAI, the photoacoustic signal per volumetric analysis was substantially higher and better visualized with Teic-ICG compared with CDX-ICG and ICG alone, and colocalized well with bioluminescence and fluorescence imaging. CONCLUSION: Photoacoustic imaging successfully localized PJI in this proof-of-concept study and demonstrates potential for clinical translation in orthopaedics.

Engineered Trehalose Permeable to Mammalian Cells.

Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre). Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants) reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.

A biocompatible "split luciferin" reaction and its application for non-invasive bioluminescent imaging of protease activity in living animals.

The great complexity of many human pathologies, such as cancer, diabetes, and neurodegenerative diseases, requires new tools for studies of biological processes on the whole organism level. The discovery of novel biocompatible reactions has tremendously advanced our understanding of basic biology; however, no efficient tools exist for real-time non-invasive imaging of many human proteases that play very important roles in multiple human disorders. We recently reported that the "split luciferin" biocompatible reaction represents a valuable tool for evaluation of protease activity directly in living animals using bioluminescence imaging (BLI). Since BLI is the most sensitive in vivo imaging modality known to date, this method can be widely applied for the evaluation of the activity of multiple proteases, as well as identification of their new peptide-specific substrates. In this unit, we describe several applications of this "split luciferin" reaction for quantification of protease activities in test tube assays and living animals.

Bioorthogonal approach to identify unsuspected drug targets in live cells.

A proteomics method to pull down secondary drug targets from live cells is described. The drug of interest is modified with trans-cyclooctene (TCO) and incubated with live cells. Upon cell lysis, the modified drug bound to the protein is pulled down using magnetic beads decorated with a cleavable tetrazine-modified linker. Samples are then run on an SDS-PAGE gel and isolated bands are submitted for mass spectrometry analysis to identify drug targets.

µHall chip for sensitive detection of bacteria.

Sensitive, rapid and phenotype-specific enumeration of pathogens is essential for the diagnosis of infectious disease, monitoring of food chains, and for defense against bioterrorism. Microbiological culture and genotyping, techniques that sensitively and selectively detect bacteria in laboratory settings, have limited application in clinical environments due to high cost, slow response times, and the need for specially trained staff and laboratory infrastructure. To address these challenges, we developed a microfluidic chip-based micro-Hall (µHall) platform capable of measuring single, magnetically tagged bacteria directly in clinical specimens with minimal sample processing. We demonstrated the clinical utility of the µHall chip by enumerating Gram-positive bacteria. The overall detection limit of the system was similar to that of culture tests (~10 bacteria), but the assay time was 50-times faster. This low-cost, single-cell analytical technique is especially well-suited to diagnose infectious diseases in resource-limited clinical settings.

A magnetic Gram stain for bacterial detection.

Magnetizing: Bacteria are often classified into gram-positive and gram-negative strains by staining with crystal violet (CV). The described bioorthogonal modification of CV with trans-cyclooctene (TCO) can be used to render gram-positive bacteria magnetic with tetrazine-functionalized magnetic nanoparticles (MNP-Tz). This method allows class-specific automated magnetic detection and magnetic separation.

A FRET-based probe with a chemically deactivatable quencher.

A new concept of a chemically deactivatable quencher is proposed for a FRET-based probe that turns-on its fluorescence by either an enzymatic cleavage or a chemical reagent (sodium dithionite). This concept allowed us to quantify the caspase-3 cleavage activity in solution and to reveal unreacted probes in cell experiments.

Ubiquitous detection of gram-positive bacteria with bioorthogonal magnetofluorescent nanoparticles.

The ability to rapidly diagnose gram-positive pathogenic bacteria would have far reaching biomedical and technological applications. Here we describe the bioorthogonal modification of small molecule antibiotics (vancomycin and daptomycin), which bind to the cell wall of gram-positive bacteria. The bound antibiotics conjugates can be reacted orthogonally with tetrazine-modified nanoparticles, via an almost instantaneous cycloaddition, which subsequently renders the bacteria detectable by optical or magnetic sensing. We show that this approach is specific, selective, fast and biocompatible. Furthermore, it can be adapted to the detection of intracellular pathogens. Importantly, this strategy enables detection of entire classes of bacteria, a feat that is difficult to achieve using current antibody approaches. Compared to covalent nanoparticle conjugates, our bioorthogonal method demonstrated 1-2 orders of magnitude greater sensitivity. This bioorthogonal labeling method could ultimately be applied to a variety of other small molecules with specificity for infectious pathogens, enabling their detection and diagnosis.