Absorption reconstruction improves biodistribution assessment of fluorescent nanoprobes using hybrid Fluorescence-mediated tomography

Aim: Fluorescence-mediated tomography (FMT) holds potential for accelerating diagnostic and theranostic drug development. However, for proper quantitative fluorescence reconstruction, knowledge on optical scattering and absorption, which are highly heterogeneous in different (mouse) tissues, is required. We here describe methods to assess these parameters using co-registered micro Computed Tomography (µCT) data and nonlinear whole-animal absorption reconstruction, and evaluate their importance for assessment of the biodistribution and target site accumulation of fluorophore-labeled drug delivery systems.\ud \ud Methods: Besides phantoms with varying degrees of absorption, mice bearing A431 tumors were imaged 15 min and 48 h after i.v. injection of a fluorophore-labeled polymeric drug carrier (pHPMA-Dy750) using µCT-FMT. The outer shape of mice and a scattering map were derived using automated segmentation of the µCT data. Furthermore, a 3D absorption map was reconstructed from the trans-illumination data. We determined the absorption of five interactively segmented regions (heart, liver, kidney, muscle, tumor). Since blood is the main near-infrared absorber in vivo, the absorption was also estimated from the relative blood volume (rBV), determined by contrast-enhanced µCT. We compared the reconstructed absorption with the rBV-based values and analyzed the effect of using the absorption map on the fluorescence reconstruction.\ud \ud Results: Phantom experiments demonstrated that absorption reconstruction is possible and necessary for quantitative fluorescence reconstruction. In vivo, the reconstructed absorption showed high values in strongly blood-perfused organs such as the heart, liver and kidney. The absorption values correlated strongly with the rBV-based absorption values, confirming the accuracy of the absorption reconstruction. Usage of homogenous absorption instead of the reconstructed absorption map resulted in reduced values in the heart, liver and kidney, by factors of 3.5, 2.1 and 1.4, respectively. For muscle and subcutaneous tumors, which have a much lower rBV and absorption, absorption reconstruction was less important.\ud \ud Conclusion: Quantitative whole-animal absorption reconstruction is possible and can be validated in vivo using the rBV. Usage of an absorption map is important when quantitatively assessing the biodistribution of fluorescently labeled drugs and drug delivery systems, to avoid a systematic underestimation of fluorescence in strongly absorbing organs, such as the heart, liver and kidney

What scans we will read: imaging instrumentation trends in clinical oncology

Oncological diseases account for a significant portion of the burden on public healthcare systems with associated costs driven primarily by complex and long-lasting therapies. Through the visualization of patient-specific morphology and functional-molecular pathways, cancerous tissue can be detected and characterized non- invasively, so as to provide referring oncologists with essential information to support therapy management decisions. Following the onset of stand-alone anatomical and functional imaging, we witness a push towards integrating molecular image information through various methods, including anato-metabolic imaging (e.g., PET/ CT), advanced MRI, optical or ultrasound imaging. This perspective paper highlights a number of key technological and methodological advances in imaging instrumentation related to anatomical, functional, molecular medicine and hybrid imaging, that is understood as the hardware-based combination of complementary anatomical and molecular imaging. These include novel detector technologies for ionizing radiation used in CT and nuclear medicine imaging, and novel system developments in MRI and optical as well as opto-acoustic imaging. We will also highlight new data processing methods for improved non-invasive tissue characterization. Following a general introduction to the role of imaging in oncology patient management we introduce imaging methods with well-defined clinical applications and potential for clinical translation. For each modality, we report first on the status quo and point to perceived technological and methodological advances in a subsequent status go section. Considering the breadth and dynamics of these developments, this perspective ends with a critical reflection on where the authors, with the majority of them being imaging experts with a background in physics and engineering, believe imaging methods will be in a few years from now. Overall, methodological and technological medical imaging advances are geared towards increased image contrast, the derivation of reproducible quantitative parameters, an increase in volume sensitivity and a reduction in overall examination time. To ensure full translation to the clinic, this progress in technologies and instrumentation is complemented by progress in relevant acquisition and image-processing protocols and improved data analysis. To this end, we should accept diagnostic images as “data”, and – through the wider adoption of advanced analysis, including machine learning approaches and a “big data” concept – move to the next stage of non-invasive tumor phenotyping. The scans we will be reading in 10 years from now will likely be composed of highly diverse multi- dimensional data from multiple sources, which mandate the use of advanced and interactive visualization and analysis platforms powered by Artificial Intelligence (AI) for real-time data handling by cross-specialty clinical experts with a domain knowledge that will need to go beyond that of plain imaging

Recent progress in nanomedicine: therapeutic, diagnostic and theranostic applications

In recent years, the use of nanomedicine formulations for therapeutic and diagnostic applications has increased exponentially. Many different systems and strategies have been developed for drug targeting to pathological sites, as well as for visualizing and quantifying important (patho-) physiological processes. In addition, ever more efforts have been undertaken to combine diagnostic and therapeutic properties within a single nanomedicine formulation. These so-called nanotheranostics are able to provide valuable information on drug delivery, drug release and drug efficacy, and they are considered to be highly useful for personalizing nanomedicine-based (chemo-) therapeutic intervention

Noninvasive optical imaging of nanomedicine biodistribution

Nanomedicines are sub-micrometer-sized carrier materials designed to improve the biodistribution of i.v. administered (chemo-) therapeutic agents. In recent years, ever more efforts in the nanomedicine field have employed optical imaging (OI) techniques to monitor biodistribution and target site accumulation. Thus far, however, the longitudinal assessment of nanomedicine biodistribution using OI has been impossible, due to limited light penetration (in the case of 2D fluorescence reflectance imaging; FRI) and to the inability to accurately allocate fluorescent signals to nonsuperficial organs (in the case of 3D fluorescence molecular tomography; FMT). Using a combination of high-resolution microcomputed tomography (μCT) and FMT, we have here set out to establish a hybrid imaging protocol for noninvasively visualizing and quantifying the accumulation of near-infrared fluorophore-labeled nanomedicines in tissues other than superficial tumors. To this end, HPMA-based polymeric drug carriers were labeled with Dy750, their biodistribution and tumor accumulation were analyzed using FMT, and the resulting data sets were fused with anatomical μCT data sets in which several different physiologically relevant organs were presegmented. The robustness of 3D organ segmentation was validated, and the results obtained using 3D CT-FMT were compared to those obtained upon standard 3D FMT and 2D FRI. Our findings convincingly demonstrate that combining anatomical μCT with molecular FMT facilitates the noninvasive assessment of nanomedicine biodistributio

Characterizing EPR-mediated passive drug targeting using contrast-enhanced functional ultrasound imaging

The Enhanced Permeability and Retention (EPR) effect is extensively used in drug delivery research. Taking into account that EPR is a highly variable phenomenon, we have here set out to evaluate if contrast-enhanced functional ultrasound (ceUS) imaging can be employed to characterize EPR-mediated passive drug targeting to tumors. Using standard fluorescence molecular tomography (FMT) and two different protocols for hybrid computed tomography-fluorescence molecular tomography (CT-FMT), the tumor accumulation of a ~ 10 nm-sized near-infrared-fluorophore-labeled polymeric drug carrier (pHPMA-Dy750) was evaluated in CT26 tumor-bearing mice. In the same set of animals, two different ceUS techniques (2D MIOT and 3D B-mode imaging) were employed to assess tumor vascularization. Subsequently, the degree of tumor vascularization was correlated with the degree of EPR-mediated drug targeting. Depending on the optical imaging protocol used, the tumor accumulation of the polymeric drug carrier ranged from 5 to 12% of the injected dose. The degree of tumor vascularization, determined using ceUS, varied from 4 to 11%. For both hybrid CT-FMT protocols, a good correlation between the degree of tumor vascularization and the degree of tumor accumulation was observed, within the case of reconstructed CT-FMT, correlation coefficients of ~ 0.8 and p-values of < 0.02. These findings indicate that ceUS can be used to characterize and predict EPR, and potentially also to pre-select patients likely to respond to passively tumor-targeted nanomedicine treatments

Micro-CT imaging of tumor angiogenesis: Quantitative measures describing micromorphology and vascularization

Angiogenesis is a hallmark of cancer, and its noninvasive visualization and quantification are key factors for facilitating translational anticancer research. Using four tumor models characterized by different degrees of aggressiveness and angiogenesis, we show that the combination of functional in vivo and anatomical ex vivo X-ray micro-computed tomography (μCT) allows highly accurate quantification of relative blood volume (rBV) and highly detailed three-dimensional analysis of the vascular network in tumors. Depending on the tumor model, rBV values determined using in vivo μCT ranged from 2.6% to 6.0%, and corresponds well with the values assessed using IHC. Using ultra–high-resolution ex vivo μCT, blood vessels as small as 3.4 μm and vessel branches up to the seventh order could be visualized, enabling a highly detailed and quantitative analysis of the three-dimensional micromorphology of tumor vessels. Microvascular parameters such as vessel size and vessel branching correlated very well with tumor aggressiveness and angiogenesis. In rapidly growing and highly angiogenic A431 tumors, the majority of vessels were small and branched only once or twice, whereas in slowly growing A549 tumors, the vessels were much larger and branched four to seven times. Thus, we consider that combining highly accurate functional with highly detailed anatomical μCT is a useful tool for facilitating high-throughput, quantitative, and translational (anti-) angiogenesis and antiangiogenesis research

Fluorescently labeled microbubbles for facilitating translational molecular ultrasound studies

Abstract Microbubbles (MB) are routinely used as contrast agents for functional and molecular ultrasound (US) imaging. For molecular US imaging, MB are functionalized with antibodies or peptides, in order to visualize receptor expression by angiogenic or inflamed endothelium. In general, initial in vitro binding studies with targeted MB are performed using phase contrast microscopy. Difficulties in the identification of MB in standard phase contrast microscopy, however, generally result in high variability, high observer dependency, and low reproducibility. To overcome these shortcomings, we here describe a simple post-loading strategy for labeling polymer-based MB with fluorophores, and we show that the use of rhodamine-loaded MB in combination with fluorescence microscopy substantially reduces the variability and the observer dependency of in vitro binding studies. In addition, we demonstrate that rhodamine-loaded MB can also be used for in vivo and ex vivo experimental setups, e.g., for analyzing MB binding to inflamed carotids using two-photon laser scanning microscopy, and for validating the binding of VEGFR2-targeted MB to tumor endothelium. These findings demonstrate that fluorescently labeled MB substantially facilitate translational molecular US studies, and they suggest that a similar synthetic strategy can be exploited for preparing drug-loaded MB, to enable image-guided, targeted, and triggered drug delivery to tumors and to sites of inflammation

Complete regression of xenograft tumors upon targeted delivery of paclitaxel via Π-Π stacking stabilized polymeric micelles

Treatment of cancer patients with taxane-based chemotherapeutics, such as paclitaxel (PTX), is complicated by their narrow therapeutic index. Polymeric micelles are attractive nanocarriers for tumor-targeted delivery of PTX, as they can be tailored to encapsulate large amounts of hydrophobic drugs and achiv prolonged circulation kinetics. As a result, PTX deposition in tumors is increased, while drug exposure to healthy tissues is reduced. However, many PTX-loaded micelle formulations suffer from low stability and fast drug release in the circulation, limiting their suitability for systemic drug targeting. To overcome these limitations, we have developed PTX-loaded micelles which are stable without chemical cross-linking and covalent drug attachment. These micelles are characterized by excellent loading capacity and strong drug retention, attributed to π–π stacking interaction between PTX and the aromatic groups of the polymer chains in the micellar core. The micelles are based on methoxy poly(ethylene glycol)-b-(N-(2-benzoyloxypropyl)methacrylamide) (mPEG-b-p(HPMAm-Bz)) block copolymers, which improved the pharmacokinetics and the biodistribution of PTX, and substantially increased PTX tumor accumulation (by more than 2000%; as compared to Taxol or control micellar formulations). Improved biodistribution and tumor accumulation were confirmed by hybrid μCT-FMT imaging using near-infrared labeled micelles and payload. The PTX-loaded micelles were well tolerated at different doses, while they induced complete tumor regression in two different xenograft models (i.e., A431 and MDA-MB-468). Our findings consequently indicate that π–π stacking-stabilized polymeric micelles are promising carriers to improve the delivery of highly hydrophobic drugs to tumors and to increase their therapeutic index

PEG-pHPMAm-based polymeric micelles loaded with doxorubicin-prodrugs in combination antitumor therapy with oncolytic vaccinia viruses

An enzymatically activatable prodrug of doxorubicin was covalently coupled, using click-chemistry, to the hydrophobic core of poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl)-methacrylamide-lactate] micelles. The release and cytotoxic activity of the prodrug was evaluated in vitro in A549 non-small-cell lung cancer cells after adding β-glucuronidase, an enzyme which is present intracellularly in lysosomes and extracellularly in necrotic areas of tumor lesions. The prodrug-containing micelles alone and in combination with standard and β-glucuronidase-producing oncolytic vaccinia viruses were also evaluated in vivo, in mice bearing A549 xenograft tumors. When combined with the oncolytic viruses, the micelles completely blocked tumor growth. Moreover, a significantly better antitumor efficacy as compared to virus treatment alone was observed when β-glucuronidase virus treated tumor-bearing mice received the prodrug-containing micelles. These findings show that combining tumor-targeted drug delivery systems with oncolytic vaccinia viruses holds potential for improving anticancer therapy