Despite intensive research and medical advances met, metastatic disease remains the most common cause of death in cancer patients. This results from late diagnosis, poor therapeutic response and undetected micrometastases and tumor margins during surgery. One approach to overcome these challenges involves fluorescence imaging, which exploits the properties of fluorescent probes for diagnostic detection of molecular structures at the onset of transformation and for intraoperative detection of metastases and tumor margins in real time. Considering these benefits, many contrast agents suitable for fluorescence imaging have been reported. However, most reports only demonstrate the detection of primary tumors and not the detection of metastases or their application in models of image-guided surgery. In this work, we demonstrate the influence of fibroblast activation protein (FAP) on the metastatic potential of fibrosarcoma cells and elucidate the efficacy of activatable FAP-targeting immunoliposomes (FAP-IL) for image-guided detection of the spontaneous metastases in mice models. Furthermore, we characterized the biodistribution and cellular localization of the liposomal fluorescent components in mice organs and traced their excretion over time in urine and feces. Taken together, activatable FAP-IL enhances intraoperative imaging of metastases. Their high accumulation in metastases, subsequent localization in the bile canaliculi and liver kupffer cells and suitable excretion in feces substantiates their potency as contrast agents for intraoperative imaging.

Molecular targeting plays a significant role in cancer diagnosis and therapy. However, the heterogeneity of tumors is a limiting obstacle for molecular targeting. Consequently, clinically approved drug delivery systems such as liposomes still rely on passive targeting to tumors, which does not address tumor heterogeneity. In this work, we therefore designed and elucidated the potentials of activatable bispecific targeted liposomes for simultaneous detection of fibroblast activation protein (FAP) and the human epidermal growth factor receptor 2 (HER2). The bispecific liposomes were encapsulated with fluorescence-quenched concentrations of the near-infrared fluorescent dye, DY-676-COOH, making them detectable solely post processing within target cells. The liposomes were endowed with a combination of single chain antibody fragments specific for FAP and HER2 respectively, or with the FAP single chain antibody fragment in combination with Trastuzumab, which is specific for HER2. The Trastuzumab based bispecific formulation, termed Bi-FAP/Tras-IL revealed delivery of the encapsulated dye into the nuclei of HER2 expressing cancer cells and caused cell death at significantly higher rates than the free Trastuzumab. Furthermore, fluorescence imaging and live microscopy of tumor models in mice substantiated the delivery of the encapsulated cargo into the nuclei of target tumor cells and tumor stromal fibroblasts. Hence, they convey potentials to address tumor plasticity, to improve targeted cancer therapy and reduce Trastuzumab resistance in the future. This work demonstrates the design of activatable bispecific liposomes aimed to target HER2, a poor prognosis tumor marker in many tumor types, and fibroblast activation protein (FAP), a universal tumor marker overexpressed on tumor fibroblasts and pericytes of almost all solid tumors. Encapsulating liposomes with a quenched concentration of a NIRF dye which only fluoresced after cellular degradation and activation enabled reliable visualization of the destination of the cargo in cells and animal studies. Conjugating single chain antibody fragments directed to FAP, together with Trastuzumab, a humanized monoclonal antibody for HER2 resulted in the activatable bispecific liposomes. In animal models of xenografted human breast tumors, the remarkable ability of the bispecific probes to simultaneously deliver the encapsulated dye into the nuclei of target tumor cells and tumor fibroblasts could be demonstrated. Hence, the bispecific probes represent model tools with high significance to address tumor heterogeneity and manage Trastuzumab resistance in the future.

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.

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. 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. 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.

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. 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. 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. 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. The endoglin-targeting liposomes presented herewith represent strategic tools for the future implementation of endoglin-directed neoplastic and anti-angiogenic therapies.

Liposomes represent suitable tools for the diagnosis and treatment of a variety of diseases, including cancers. To study the role of the human epidermal growth factor receptor 2 (HER2) as target in cancer imaging and image-guided deliveries, liposomes were encapsulated with an intrinsically quenched concentration of a near-infrared fluorescent dye in their aqueous interior. This resulted in quenched liposomes (termed LipQ), that were fluorescent exclusively upon degradation, dye release, and activation. The liposomes carried an always-on green fluorescent phospholipid in the lipid layer to enable tracking of intact liposomes. Additionally, they were functionalized with single-chain antibody fragments directed to fibroblast activation protein (FAP), a marker of stromal fibroblasts of most epithelial cancers, and to HER2, whose overexpression in 20–30% of all breast cancers and many other cancer types is associated with a poor treatment outcome and relapse. We show that both monospecific (HER2-IL) and bispecific (Bi-FAP/HER2-IL) formulations are quenched and undergo HER2-dependent rapid uptake and cargo release in cultured target cells and tumor models in mice. Thereby, tumor fluorescence was retained in whole-body NIRF imaging for 32–48 h post-injection. Opposed to cell culture studies, Bi-FAP/HER2-IL-based live confocal microscopy of a high HER2-expressing tumor revealed nuclear delivery of the encapsulated dye. Thus, the liposomes have potentials for image-guided nuclear delivery of therapeutics, and also for intraoperative delineation of tumors, metastasis, and tumor margins.