Project description:Current drug-safety assays for hepatotoxicity rely on biomarkers with low predictive power. The production of radical species, specifically reactive oxygen species (ROS) and reactive nitrogen species (RNS), has been proposed as an early unifying event linking the bioactivation of drugs to hepatotoxicity and as a more direct and mechanistic indicator of hepatotoxic potential. Here we present a nanosensor for rapid, real-time in vivo imaging of drug-induced ROS and RNS for direct evaluation of acute hepatotoxicity. By combining fluorescence resonance energy transfer (FRET) and chemiluminescence resonance energy transfer (CRET), our semiconducting polymer-based nanosensor simultaneously and differentially detects RNS and ROS using two optically independent channels. We imaged drug-induced hepatotoxicity and its remediation longitudinally in mice after systemic challenge with acetaminophen or isoniazid. We detected dose-dependent ROS and RNS activity in the liver within minutes of drug challenge, which preceded histological changes, protein nitration and DNA double-strand-break induction.
Project description:Worldwide, tuberculosis (TB) is the leading cause of death due to infection with a single pathogenic agent, Mycobacterium tuberculosis In the absence of an effective vaccine, new, more powerful antibiotics are required to halt the growing spread of multidrug-resistant strains and to shorten the duration of TB treatment. However, assessing drug efficacy at the preclinical stage remains a long and fastidious procedure that delays progression of drugs down the pipeline and towards the clinic. In this investigation, we report the construction, optimization and characterization of genetically engineered near-infrared (NIR) fluorescent reporter strains of the pathogens Mycobacterium marinum and Mycobacterium tuberculosis that enable direct visualization of bacteria in infected zebrafish and mice, respectively. Fluorescence could be measured precisely in infected immunodeficient mice, while its intensity appeared to be below the limit of detection in immunocompetent mice, probably because of the lower bacterial load obtained in these animals. Furthermore, we show that the fluorescence level accurately reflects the bacterial load, as determined by colony forming unit (CFU) enumeration, thus enabling the efficacy of antibiotic treatment to be assessed in live animals in real time. The NIR fluorescent imaging system disclosed here is a valuable resource for TB research and can serve to accelerate drug development.
Project description:Disseminated tumor cells in the bone marrow environment are the main cause of systemic metastasis after curative treatment for major solid tumors. However, the detailed biological processes of tumor biology in bone marrow have not been well defined in a real-time manner, because of a lack of a proper in vivo experimental model thereof. In this study, we established intravital imaging models of the bone marrow environment to enable real-time observation of cancer cells in the bone marrow. Using these novel imaging models of intact bone marrow and transplanted bone marrow of mice, respectively, via two-photon microscopy, we could first successfully track and analyze both the distribution and the phenotype of cancer cells in bone marrow of live mouse. Therefore, these novel in vivo imaging models for the bone marrow would provide a valuable tool to identify the biologic processes of cancer cells in a real-time manner in a live animal model.
Project description:Tuberculosis (TB) remains a leading cause of death worldwide. Lipid rich, phenotypically antibiotic tolerant, bacteria are more resistant to antibiotics and may be responsible for relapse and the need for long-term TB treatment. We present a microfluidic system that acoustically traps live mycobacteria, M. smegmatis, a model organism for M. tuberculosis. We then perform optical analysis in the form of wavelength modulated Raman spectroscopy (WMRS) on the trapped M. smegmatis for up to eight hours, and also in the presence of isoniazid (INH). The Raman fingerprints of M. smegmatis exposed to INH change substantially in comparison to the unstressed condition. Our work provides a real-time assessment of the impact of INH on the increase of lipids in these mycobacteria, which could render the cells more tolerant to antibiotics. This microfluidic platform may be used to study any microorganism and to dynamically monitor its response to different conditions and stimuli.
Project description:Tissue-engineered in vitro models have the potential to be used for investigating inflammation in the complex microenvironment found in vivo. We have developed an in vitro model of hepatic tissue that facilitates real-time monitoring of endothelium activation in liver tissue. This was achieved by creating a layered coculture model in which hepatocytes were embedded in collagen gel and a reporter clone of endothelial cells, which synthesizes green fluorescent protein in response to nuclear factor-kappa B (NF-κB) activation, was overlaid on top of the gel. The efficacy of our approach was established by monitoring in real time the dynamics of NF-κB-regulated fluorescence in response to tumor necrosis factor α. Our studies revealed that endothelial cells in coculture with hepatocytes exhibited a similar NF-κB-mediated fluorescence to both pulse and step stimulation of lipopolysaccharide. By contrast, endothelial cells in monoculture displayed enhanced NF-κB-regulated fluorescence to step in comparison to pulse lipopolysaccharide stimulation. The NF-κB-mediated fluorescence correlated with endothelial cell expression of NF-κB-regulated genes such as intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and E-Selectin, as well as with leukocyte adhesion. These findings suggest that our model provides a powerful platform for investigating hepatic endothelium activation in real time.
Project description:Biosensors that continuously measure circulating biomolecules in real time could provide insights into the health status of patients and their response to therapeutics. But biosensors for the continuous real-time monitoring of analytes in vivo have only reached nanomolar sensitivity and can measure only a handful of molecules, such as glucose and blood oxygen. Here we show that multiple analytes can be continuously and simultaneously measured with picomolar sensitivity and sub-second resolution via the integration of aptamers and antibodies into a bead-based fluorescence sandwich immunoassay implemented in a custom microfluidic chip. After an incubation time of 30 s, bead fluorescence is measured using a high-speed camera under spatially multiplexed two-colour laser illumination. We used the assay for continuous quantification of glucose and insulin concentrations in the blood of live diabetic rats to resolve inter-animal differences in the pharmacokinetic response to insulin as well as discriminate pharmacokinetic profiles from different insulin formulations. The assay can be readily modified to continuously and simultaneously measure other blood analytes in vivo.
Project description:Background and purposeIn this paper, we investigate the possibility to use X-ray based real time 2D/3D registration for non-invasive tumor motion monitoring during radiotherapy.Materials and methodsThe 2D/3D registration scheme is implemented using general purpose computation on graphics hardware (GPGPU) programming techniques and several algorithmic refinements in the registration process. Validation is conducted off-line using a phantom and five clinical patient data sets. The registration is performed on a region of interest (ROI) centered around the planned target volume (PTV).ResultsThe phantom motion is measured with an rms error of 2.56 mm. For the patient data sets, a sinusoidal movement that clearly correlates to the breathing cycle is shown. Videos show a good match between X-ray and digitally reconstructed radiographs (DRR) displacement. Mean registration time is 0.5 s.ConclusionsWe have demonstrated that real-time organ motion monitoring using image based markerless registration is feasible.
Project description:Magnetic drug targeting has been proposed as means of concentrating therapeutic agents at a target site and the success of this approach has been demonstrated in a number of studies. However, the behavior of magnetic carriers in blood vessels and tumor microcirculation still remains unclear. In this work, we utilized polymeric magnetic nanocapsules (m-NCs) for magnetic targeting in tumors and dynamically visualized them within blood vessels and tumor tissues before, during and after magnetic field exposure using fibered confocal fluorescence microscopy (FCFM). Our results suggested that the distribution of m-NCs within tumor vasculature changed dramatically, but in a reversible way, upon application and removal of a magnetic field. The m-NCs were concentrated and stayed as clusters near a blood vessel wall when tumors were exposed to a magnetic field but without rupturing the blood vessel. The obtained FCFM images provided in vivo in situ microvascular observations of m-NCs upon magnetic targeting with high spatial resolution but minimally invasive surgical procedures. This proof-of-concept descriptive study in mice is envisaged to track and quantify nanoparticles in vivo in a non-invasive manner at microscopic resolution.
Project description:Molecular photoacoustic imaging (PA) is a promising technology to understand tumor pathology and guide precision therapeutics. Despite the capability of activatable PA probes to image tumor-specific biomarkers, limitations in their molecular structure hamper them from effective drug delivery and the drug release monitoring. Herein, we developed a perylene diimide (PDI) based theranostic platform that provides noninvasive PA imaging signals to monitor tumor-specific pH-responsive drug release. Methods: we first designed and synthesized an acid-responsive amine-substituted PDI derivative. The pH sensitive properties of the PDI was demonstrated by density functional theory (DFT) calculations, UV-vis experiments and PA studies. The theranostic platform (THPDINs) was fabricated by self-assembly of the acid-responsive PDI, a pH irrelevant IR825 dye, and anti-cancer drug doxorubicin (DOX). The PA properties in various pH environment, drug delivery, cytotoxicity, cell uptake, ratiometric PA imaging and anti-tumor efficacy of the THPDINs were investigated in vitro and in vivo by using U87MG glioma cell line and U87MG tumor model. Results: We found that our designed PDI was sensitive to the tumor specific pH environment, reflected by absorbance shift, PA intensity and aggregation morphology changes in aqueous solution. The as-synthesized pH sensitive PDI acted as a molecular switch in the THPDINs, in which the switch can be triggered in the mild acidic tumor microenvironment to accelerate DOX release. Meanwhile, the DOX release could be monitored by ratiometric PA imaging. Conclusions: We developed a multifunctional PDI based theranostic platform for noninvasive real-time ratiometric PA imaging of tumor acidic pH and monitoring of drug release in living mice simultaneously. This strategy will shed light on the development of smart activatable theranostic nanoplatforms and will significantly advance the application of PA theranostics in biology and medicine.
Project description:Treatment of MCF7 breast cancer cells by cisplatin leads to a very specific metabolic response and an onset of cell death about 10-11 h after beginning of treatment. For more detailed understanding of the molecular processes underlying the specific metabolic response, mRNA was isolated from MCF7 cells when the specific changes, (i) induction of glycolysis and (ii) onset of cell death, were detected during online measurement in the cell biosensor system.