Endoglin based in vivo near-infrared fluorescence imaging of tumor models in mice using activatable liposomes.

BACKGROUND: Endoglin (CD105) is overexpressed on tumor cells and tumor vasculatures, making it a potential target for diagnostic imaging and therapy of different neoplasms. Therefore, studies on nanocarrier systems designed for endoglin-directed diagnostic and drug delivery purposes would expose the feasibility of targeting endoglin with therapeutics. METHODS: Liposomes carrying high concentrations of a near-infrared fluorescent dye in the aqueous interior were prepared by the lipid film hydration and extrusion procedure, then conjugated to single chain antibody fragments either selective for murine endoglin (termed mEnd-IL) or directed towards human endoglin (termed hEnd-IL). A combination of Dynamic Light Scattering, electron microscopy, cell binding and uptake assays, confocal microscopy and in vivo fluorescence imaging of mice bearing xenografted human breast cancer and human fibrosarcoma models were implemented to elucidate the potentials of the liposomes. RESULTS: The mEnd-IL and hEnd-IL were highly selective for the respective murine- and human endoglin expressing cells in vitro and in vivo. Hence, the hEnd-IL bound distinctly to the tumor cells and enabled suitable fluorescence imaging of the tumors, whereas the mEnd-IL bound the tumor vasculature, but also to the liver, kidney and lung vasculature of mice. CONCLUSIONS: The work highlights key differences between targeting vascular (murine) and neoplastic (human) endoglin in animal studies, and suggests that the hEnd-IL can serve as a delivery system that targets human endoglin overexpressed in pathological conditions. GENERAL SIGNIFICANCE: The endoglin-targeting liposomes presented herewith represent strategic tools for the future implementation of endoglin-directed neoplastic and anti-angiogenic therapies.

A fast and effective determination of the biodistribution and subcellular localization of fluorescent immunoliposomes in freshly excised animal organs.

BACKGROUND: Preclinical research implementing fluorescence-based approaches is inevitable for drug discovery and technology. For example, a variety of contrast agents developed for biomedical imaging are usually evaluated in cell systems and animal models based on their conjugation to fluorescent dyes. Biodistribution studies of excised organs are often performed by macroscopic imaging, whereas the subcellular localization though vital, is often neglected or further validated by histological procedures. Available systems used to define the subcellular biodistribution of contrast agents such as intravital microscopes or ex vivo histological analysis are expensive and not affordable by the majority of researchers, or encompass tedious and time consuming steps that may modify the contrast agents and falsify the results. Thus, affordable and more reliable approaches to study the biodistribution of contrast agents are required. We developed fluorescent immunoliposomes specific for human fibroblast activation protein and murine endoglin, and used macroscopic fluorescence imaging and confocal microscopy to determine their biodistribution and subcellular localization in freshly excised mice organs at different time points post intravenous injection. RESULTS: Near infrared fluorescence macroscopic imaging revealed key differences in the biodistribution of the respective immunoliposomes at different time points post injection, which correlated to the first-pass effect as well as the binding of the probes to molecular targets within the mice organs. Thus, a higher accumulation and longer retention of the murine endoglin immunoliposomes was seen in the lungs, liver and kidneys than the FAP specific immunoliposomes. Confocal microscopy showed that tissue autofluorescence enables detection of organ morphology and cellular components within freshly excised, non-processed organs, and that fluorescent probes with absorption and emission maxima beyond the tissue autofluorescence range can be easily distinguished. Hence, the endoglin targeting immunoliposomes retained in some organs could be detected in the vascular endothelia cells of the organs. CONCLUSIONS: The underlying work represents a quick, effective and more reliable setup to validate the macroscopic and subcellular biodistribution of contrast agents in freshly excised animal organs. The approach will be highly beneficial to many researchers involved in nanodrug design or in fluorescence-based studies on disease pathogenesis.

Targeting the Tumor Microenvironment with Fluorescence-Activatable Bispecific Endoglin/Fibroblast Activation Protein Targeting Liposomes.

Liposomes are biocompatible nanocarriers with promising features for targeted delivery of contrast agents and drugs into the tumor microenvironment, for imaging and therapy purposes. Liposome-based simultaneous targeting of tumor associated fibroblast and the vasculature is promising, but the heterogeneity of tumors entails a thorough validation of suitable markers for targeted delivery. Thus, we elucidated the potential of bispecific liposomes targeting the fibroblast activation protein (FAP) on tumor stromal fibroblasts, together with endoglin which is overexpressed on tumor neovascular cells and some neoplastic cells. Fluorescence-quenched liposomes were prepared by hydrating a lipid film with a high concentration of the self-quenching near-infrared fluorescent dye, DY-676-COOH, to enable fluorescence detection exclusively upon liposomal degradation and subsequent activation. A non-quenched green fluorescent phospholipid was embedded in the liposomal surface to fluorescence-track intact liposomes. FAP- and murine endoglin-specific single chain antibody fragments were coupled to the liposomal surface, and the liposomal potentials validated in tumor cells and mice models. The bispecific liposomes revealed strong fluorescence quenching, activatability, and selectivity for target cells and delivered the encapsulated dye selectively into tumor vessels and tumor associated fibroblasts in xenografted mice models and enabled their fluorescence imaging. Furthermore, detection of swollen lymph nodes during intra-operative simulations was possible. Thus, the bispecific liposomes have potentials for targeted delivery into the tumor microenvironment and for image-guided surgery.

Dataset on the role of endoglin expression on melanin production in murine melanoma and on the influence of melanin on optical imaging.

The underlying data demonstrates that the expression of endoglin in murine melanoma cells influences melanin production in the cells. Also, the data shows that melanin production is further increased when the cells are subcutaneously implanted in mice models and that the high melanin production prevents detection of the cells by fluorescence imaging. The processed data presented herein is related to a research article by Tansi et al. (2018) entitled "Endoglin based in vivo near-infrared fluorescence imaging of tumor models in mice using activatable liposomes".