Increased Depth of Cellular Imaging in the Intact Lung Using Far-Red and Near-Infrared Fluorescent Probes

Scattering of shorter-wavelength visible light limits the fluorescence imaging depth of thick specimens such as whole organs. In this study, we report the use of four newly synthesized near-infrared and far-red fluorescence probes (excitation/emission, in nm: 644/670; 683/707; 786/814; 824/834) to image tumor cells in the subpleural vasculature of the intact rat lungs. Transpelural imaging of tumor cells labeled with long-wavelength probes and expressing green fluorescent protein (GFP; excitation/emission 488/507 nm) was done in the intact rat lung after perfusate administration or intravenous injection. Our results show that the average optimum imaging depth for the long-wavelength probes is higher (27.8 ± 0.7  μm) than for GFP (20 ± 0.5  μm; p = 0.008; n = 50), corresponding to a 40% increase in the volume of tissue accessible for high-resolution imaging. The maximum depth of cell visualization was significantly improved with the novel dyes (36.4 ± 1  μm from the pleural surface) compared with GFP (30.1 ± 0.5  μm; p = 0.01; n = 50). Stable binding of the long-wavelength vital dyes to the plasma membrane also permitted in vivo tracking of injected tumor cells in the pulmonary vasculature. These probes offer a significant improvement in the imaging quality of in situ biological processes in the deeper regions of intact lungs

Optical imaging in vivo with a focus on paediatric disease: technical progress, current preclinical and clinical applications and future perspectives

To obtain information on the occurrence and location of molecular events as well as to track target-specific probes such as antibodies or peptides, drugs or even cells non-invasively over time, optical imaging (OI) technologies are increasingly applied. Although OI strongly contributes to the advances made in preclinical research, it is so far, with the exception of optical coherence tomography (OCT), only very sparingly applied in clinical settings. Nevertheless, as OI technologies evolve and improve continuously and represent relatively inexpensive and harmful methods, their implementation as clinical tools for the assessment of children disease is increasing. This review focuses on the current preclinical and clinical applications as well as on the future potential of OI in the clinical routine. Herein, we summarize the development of different fluorescence and bioluminescence imaging techniques for microscopic and macroscopic visualization of microstructures and biological processes. In addition, we discuss advantages and limitations of optical probes with distinct mechanisms of target-detection as well as of different bioluminescent reporter systems. Particular attention has been given to the use of near-infrared (NIR) fluorescent probes enabling observation of molecular events in deeper tissue

Fluorescence molecular tomography: Principles and potential for pharmaceutical research

Fluorescence microscopic imaging is widely used in biomedical research to study molecular and cellular processes in cell culture or tissue samples. This is motivated by the high inherent sensitivity of fluorescence techniques, the spatial resolution that compares favorably with cellular dimensions, the stability of the fluorescent labels used and the sophisticated labeling strategies that have been developed for selectively labeling target molecules. More recently, two and three-dimensional optical imaging methods have also been applied to monitor biological processes in intact biological organisms such as animals or even humans. These whole body optical imaging approaches have to cope with the fact that biological tissue is a highly scattering and absorbing medium. As a consequence, light propagation in tissue is well described by a diffusion approximation and accurate reconstruction of spatial information is demanding. While in vivo optical imaging is a highly sensitive method, the signal is strongly surface weighted, i.e., the signal detected from the same light source will become weaker the deeper it is embedded in tissue, and strongly depends on the optical properties of the surrounding tissue. Derivation of quantitative information, therefore, requires tomographic techniques such as fluorescence molecular tomography (FMT), which maps the three-dimensional distribution of a fluorescent probe or protein concentration. The combination of FMT with a structural imaging method such as X-ray computed tomography (CT) or Magnetic Resonance Imaging (MRI) will allow mapping molecular information on a high definition anatomical reference and enable the use of prior information on tissue’s optical properties to enhance both resolution and sensitivity. Today many of the fluorescent assays originally developed for studies in cellular systems have been successfully translated for experimental studies in animals. The opportunity of monitoring molecular processes non-invasively in the intact organism is highly attractive from a diagnostic point of view but even more so for the drug developer, who can use the techniques for proof-of-mechanism and proof-of-efficacy studies. This review shall elucidate the current status and potential of fluorescence tomography including recent advances in multimodality imaging approaches for preclinical and clinical drug development

Spectral imaging in preclinical research and clinical pathology.

Spectral imaging methods are attracting increased interest from researchers and practitioners in basic science, pre-clinical and clinical arenas. A combination of better labeling reagents and better optics creates opportunities to detect and measure multiple parameters at the molecular and cellular level. These tools can provide valuable insights into the basic mechanisms of life, and yield diagnostic and prognostic information for clinical applications. There are many multispectral technologies available, each with its own advantages and limitations. This chapter will present an overview of the rationale for spectral imaging, and discuss the hardware, software and sample labeling strategies that can optimize its usefulness in clinical settings

Improving Antibody-drug Conjugate Tumor Distribution and Efficacy Using Single-Cell Imaging and Multiscale Modeling

Antibody-drug conjugates (ADCs) are a targeted cancer therapy combining the tumor cell specificity of antibodies with small-molecule chemotherapy. Despite the widespread use of ADC therapeutics, they exhibit a heterogeneous, perivascular distribution in tumors, often leaving significant portions of the tumor untargeted. Furthermore, the relationship between the heterogeneous distribution of ADCs in tumors and their overall efficacy is poorly understood and therefore can be underappreciated. In this thesis, I develop experimental techniques to quantify ADC distribution in tumors using near-infrared (NIR) fluorophores, construct a computational model to simulate antibody distribution at several length scales, and show, for the first time, that the antibody distribution in the tumor plays an important role in the efficacy of ADCs. To better characterize the multiscale distribution of ADCs, I first measure the residualization properties of common NIR dyes, identifying both non-residualizing and residualizing dyes. Next, I show that fluorescent dye structure and dye-to-protein ratio can be optimized for labeling antibodies with NIR fluorophores to prevent the dye from impacting antibody pharmacokinetics. I then develop a novel dual label, ratio-imaging technique to quantify antibody distribution and metabolism in vivo with unprecedented single cell resolution. Using this technique, I show the clinical dose of 3.6 mg/kg the distribution of T-DM1 is heterogeneous in high HER2 expressing tumors, only targeting 10% of tumor cells. Examining the absolute uptake of ADC in targeted cells shows that they actually receive more ADC than necessary to kill the cell, despite most of the tumor not receiving any ADC. In the second part of my thesis, I develop a multiscale modeling framework combining a physiologically-based pharmacokinetic (PBPK) and Krogh Cylinder tissue model to predict both the systemic and tumoral distribution of antibodies. Using this model, I predict, and verify experimentally, that coadministration of trastuzumab with T-DM1 at 3:1 and 8:1 ratios drives a constant dose of T-DM1 deeper into the tumor. Using this dosing strategy, the total number of cells targeted increases albeit with a lower average number of ADC molecules per cell. These results are consistent across a number of antibodies, targets, and payloads, indicating the model can be used to predict ADC distribution in other tumor models. Finally, I test the efficacy of coadministration of trastuzumab with T-DM1 in a trastuzumab resistant xenograft mouse model. T-DM1 therapy alone showed a significant improvement in efficacy and survival, as expected, while trastuzumab alone had no impact. Counterintuitively, coadministration of trastuzumab, which has no efficacy in vivo and is antagonistic to T-DM1 in vitro, actually acts synergistically with T-DM1 in vivo. Coadministration of trastuzumab at 3:1 and 8:1 trastuzumab to T-DM1 dosing levels show a statistically significant improvement in survival over T-DM1 alone. These results are the first to show that the tumoral distribution of ADCs plays a major role in their overall efficacy. Overall, this dissertation provides unique tools to study antibody and ADC distribution and metabolism, quantitative computational tools to simulate in vivo distribution, and concrete guidance on how to improve efficacy of ADC therapeutics. Although I show the importance of the antibody distribution in the tumor for efficacy, additional imaging with other ADC systems, lower and more heterogeneous antigen expressing tumors, and the antibody distribution in clinical samples will further improve our understanding of the relationship between distribution and efficacy.PHDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/147665/1/ccillier_1.pd

Bioresponsive molecular imaging probes

Molecular imaging is recognized as a powerful tool to visualize and characterize biological processes at the cellular and molecular level in vivo. In most imaging approaches, molecular probes are used to bind to disease-specific biomarkers highlighting disease target sites. In recent years, a new subset of molecular imaging probes, known as bioresponsive molecular probes, has been developed. Chapter 1 reviews the several types of these activatable imaging probes and its potential in vivo applicability. The goal of this thesis was the design, synthesis, and in vitro and in vivo characterization of novel bioresponsive imaging probes based on the elegant concept of activatable cell penetrating peptides (ACPPs). The experimental part of this thesis starts with the development of radiolabeled matrix metalloproteinase-2, -9 (MMP-2/9) activatable cell penetrating peptides. The matrix metalloproteinases -2 and -9 play an important role in angiogenesis and metastasis in cancer, and in adverse cardiac remodeling after myocardial infarction. In Chapter 2, it is shown that the proposed MMP-2/9 sensitive peptide-based imaging probes were successfully synthesized on the solid phase, and could be efficiently labeled with radio-isotopes. A dual-isotope labeled ACPP is presented that could discriminate between uptake of the activated probe and the integral probe and this ACPP was used to follow the activation process in vivo. Despite the probe showed specific sensitivity towards MMP-2 and MMP-9 in vitro, in vivo studies in tumor-bearing mice demonstrated that the ACPP was not activated in MMP-expressing tumor tissue, but most likely already in the circulation. Chapter 3 describes the in vivo characterization of the radiolabeled MMP-2/9 ACPPs in a mouse model of myocardial infarction. In this model, infarct-specific activation and retention of the MMP-2/9 ACPP was observed, as was assessed by biodistribution studies using the dual-isotope labeled ACPP. A significant correlation was found between MMP-2/9 expression and the degree of probe activation in infarcted and remote areas of the hearts. Furthermore, ACPP retention in infarcted regions was successfully visualized ex vivo using autoradiography. Nevertheless, also ACPP activation in the circulation resulted in retention of the activated probe in the surrounding tissues, especially in the liver. Consequently, a strong background signal was observed. Chapter 4 focuses on the development of long circulating MMP-2/9 sensitive ACPPs to achieve an extended exposure time to the target proteases. Incorporation of two different albumin ligands i.e. palmitic acid (Palm) and deoxycholic acid (DOCA), in the ACPPs resulted in a strong increase in circulation time of these albumin-binding ACPPs compared to the ACPP without albumin ligand. In vivo biodistribution studies in a mouse model of myocardial infarction pointed towards local activation of a DOCA-conjugated ACPP in areas of cardiac remodelling. Despite the increased circulation time of this probe, the infarct-to-remote ratios and absolute probe uptake in infarcted areas of the heart was comparable to dACPP. In view of the findings discussed in Chapters 2 and 3, we hypothesized that ACPPs sensitive for tissue-specific biomarkers should exhibit reduced activation in the vasculature and background probe uptake of the activated ACPP in all tissues. Consequently, this should improve the signal-to-background ratios of these probes. Therefore, Chapter 5 and Chapter 6 are dedicated to the development of radiolabeled ACPPs activatable by membrane-type matrix metalloproteinase-1 (MMP-14), and the transmembrane protein angiotensin converting enzyme (ACE), respectively. In Chapter 5, the design and synthesis of MMP-14 sensitive ACPPs (ACPP-14) is addressed. MMP-14, like MMP-2 and -9, plays an important role in adverse cardiac remodeling. The most effective ACPP-14 probe was selected by employing MMP-14 sensitivity and enzyme specificity assays. This probe showed efficient cellular uptake upon activation. In a pilot in vivo biodistribution study, the level of in vivo background activation in the vasculature was decreased compared to MMP-2/9 ACPP (Chapters 2 and 3), while an increased uptake in infarcted heart tissue was observed compared to remote heart tissue, warranting further research into the in vivo biodistribution of this probe. Chapter 6 presents the development of an ACPP sensitive for the carboxy exopeptidase angiotensin converting enzyme (ACE). Upregulated heart-associated ACE activity has been related to adverse cardiac remodeling in nearly all cardiovascular diseases. Using molecular modeling approaches, various ACE ACPPs were designed to fit into the catalytic pocket of ACE. These probes were subsequently synthesized, but unfortunately showed no in vitro sensitivity towards ACE. Chapter 7 deals with the design of an ACPP that responds to hydrogen peroxide (H2O2). Production of reactive oxygen species like H2O2 typically occurs during elevated oxidative stress and contributes to the pathogenesis of several diseases, including cardiac ischemia-reperfusion injury. In this Chapter, we propose to extend the application of the ACPP imaging concept to the detection and imaging of H2O2 after cardiac ischemia-reperfusion injury. The suggested H2O2 ACPP uses a H2O2 self-immolative linker moiety to which the cell penetrating polycationic peptide and the polyanionic peptide are conjugated. H2O2-triggering initiates self-immolation of the linker, and thereby releases the polycationic cell penetrating peptide in H2O2-producing tissues. This H2O2 ACPP probe is currently in development. Finally, Chapter 8 concludes with a general discussion on the preceding chapters, followed by some future perspectives of activatable cell penetrating peptide imaging probe

Core-shell semiconductor nanocrystals: Effect of composition, size, surface coatings on their optical properties, toxicity, and pharmacokinetics

Quantum dots are semiconducting nanocrystals that exhibit extraordinary optical properties. QD have shown higher photostability compared to standard organic dye type probes. Therefore, they have been heavily explored in the biomedical field. This review will discuss the different approaches to synthesis, solubilise and functionalise QD. Their main biomedical applications in imaging and photodynamic therapy will be highlighted. Finally, QD biodistribution profile and in vivo toxicity will be discussed