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.

Immuno-LipoTRAIL: Targeted delivery of TRAIL-functionalized liposomal nanoparticles.

The TNF-related apoptosis-inducing ligand (TRAIL) is a powerful inducer of apoptosis in tumor cells; however, clinical studies with recombinant soluble TRAIL were rather disappointing. Here, we developed TRAIL-functionalized liposomes (LipoTRAIL, LT) to mimic membrane-displayed TRAIL for efficient activation of death receptors DR4 and DR5 and enhanced induction of apoptosis, which were combined with an anti-EGFR single-chain Fv fragment (scFv) for targeted delivery to EGFR-positive tumor cells. These immuno-LipoTRAILs (ILTs) bound specifically to EGFR-expressing cells (Colo205) and exhibited increased cytotoxicity compared with that of nontargeted LTs. Compared to that of the soluble TRAIL, the plasma half-life of the functionalized liposomes was strongly extended, and increased antitumor activity of LT and ILT was demonstrated in a xenograft tumor model. Thus, we established a multifunctional liposomal TRAIL formulation (ILT) with improved pharmacokinetic and pharmacodynamic behavior, characterized by targeted delivery and increased induction of apoptosis due to multivalent TRAIL presentation.

[FeFe]-hydrogenase models assembled into vesicular structures.

Compartmentalization is a major prerequisite for the origin of life on earth according to Wächtershäuser "Iron-Sulfur-World". The hypothesis is mainly based on an autocatalytic inorganic energy reproducing redox system consisting of iron and sulfur as requirement for the subsequent synthesis of complex organic structures. Here, we modified [FeFe]-hydrogenase models by means of covalent coupling to either oleic acid or the amphiphilic block copolymer polybutadiene-polyethyleneoxide (PB-PEO) and incorporated those into the membranes of vesicles composed of phospholipids (liposomes) or the unmodified amphiphilic polymer (polymersomes). We employed a [2Fe-2S] cluster as a hydrogenase model, since these structures are known to be suitable catalysts for the generation of H2 in the presence of weak acids. Successful incorporation was confirmed by spectrophotometric iron quantification and the vesicles formed were characterized by size determination (photon correlation spectroscopy (PCS)), and zeta potential as well as by cryo-transmission electron microscopy (Cryo-TEM). The modified models could be incorporated into liposomes or polymersomes up to molar proportions of 3.15% and 28%, respectively. Due to the immobilization in vesicular bilayers the [FeFe]-hydrogenase models can even exhibit catalytic action under the particular conditions of the intravesicular microenvironment. Our results suggest that the vesicular systems described may be applied as a nanoreactor for the reduction of encapsulated substances by generating hydrogen and thus as a minimal cell model.

Liposomal encapsulation of a near-infrared fluorophore enhances fluorescence quenching and reliable whole body optical imaging upon activation in vivo.

In the past decade, there has been significant progress in the development of water soluble near-infrared fluorochromes for use in a wide range of imaging applications. Fluorochromes with high photo and thermal stability, sensitivity, adequate pharmacological properties and absorption/emission maxima within the near infrared window (650-900 nm) are highly desired for in vivo imaging, since biological tissues show very low absorption and auto-fluorescence at this spectrum window. Taking these properties into consideration, a myriad of promising near infrared fluorescent probes has been developed recently. However, a hallmark of most of these probes is a rapid clearance in vivo, which hampers their application. It is hypothesized that encapsulation of the near infrared fluorescent dye DY-676-COOH, which undergoes fluorescence quenching at high concentrations, in the aqueous interior of liposomes will result in protection and fluorescence quenching, which upon degradation by phagocytes in vivo will lead to fluorescence activation and enable imaging of inflammation. Liposomes prepared with high concentrations of DY-676-COOH reveal strong fluorescence quenching. It is demonstrated that the non-targeted PEGylated fluorescence-activatable liposomes are taken up predominantly by phagocytosis and degraded in lysosomes. Furthermore, in zymosan-induced edema models in mice, the liposomes are taken up by monocytes and macrophages which migrate to the sites of inflammation. Opposed to free DY-676-COOH, prolonged stability and retention of liposomal-DY-676-COOH is reflected in a significant increase in fluorescence intensity of edema. Thus, protected delivery and fluorescence quenching make the DY-676-COOH-loaded liposomes a highly promising contrast agent for in vivo optical imaging of inflammatory diseases.

Antimicrobial peptide-modified liposomes for bacteria targeted delivery of temoporfin in photodynamic antimicrobial chemotherapy.

Photodynamic antimicrobial chemotherapy (PACT) and antimicrobial peptides (AMPs) are two promising strategies to combat the increasing prevalence of antibiotic-resistant bacteria. To take advantage of these two strategies, we integrated a novel antimicrobial peptide (WLBU2) and a potent generation II photosensitizer (temoporfin) into liposomes by preparing WLBU2-modified liposomes, aiming at bacteria targeted delivery of temoporfin for PACT. WLBU2 was successfully coupled to temoporfin-loaded liposomes using a functional phospholipid. The delivery of temoporfin to bacteria was confirmed by fluorescence microscopy and flow cytometry, thus demonstrating that more temoporfin was delivered to bacteria by WLBU2-modified liposomes than by unmodified liposomes. Consequently, the WLBU2-modified liposomes eradicated all methicillin-resistant Staphylococcus aureus (MRSA) and induced a 3.3 log(10) reduction of Pseudomonas aeruginosa in the in vitro photodynamic inactivation test. These findings demonstrate that the use of AMP-modified liposomes is promising for bacteria-targeted delivery of photosensitizers and for improving the PACT efficiency against both gram-positive and gram-negative bacteria in the local infections.

Polymersomes containing iron sulfide (FeS) as primordial cell model : for the investigation of energy providing redox reactions.

According to Wächtershäuser's "Iron-Sulfur-World" one major requirement for the development of life on the prebiotic Earth is compartmentalization. Vesicles spontaneously formed from amphiphilic components containing a specific set of molecules including sulfide minerals may have lead to the first autotrophic prebiotic units. The iron sulfide minerals may have been formed by geological conversions in the environment of deep-sea volcanos (black smokers), which can be observed even today. Wächtershäuser postulated the evolution of chemical pathways as fundamentals of the origin of life on earth. In contrast to the classical Miller-Urey experiment, depending on external energy sources, the "Iron-Sulfur-World" is based on the catalytic and energy reproducing redox system FeS+H2S-->FeS2+H2. The energy release out of this redox reaction (∆RG°=-38 kJ/mol, pH 0) could be the cause for the subsequent synthesis of complex organic molecules and the precondition for the development of more complex units similar to cells known today. Here we show the possibility for precipitating iron sulfide inside vesicles composed of amphiphilic block-copolymers as a model system for a first prebiotic unit. Our findings could be an indication for a chemoautotrophic FeS based origin of life.

Rapid Target Binding and Cargo Release of Activatable Liposomes Bearing HER2 and FAP Single-Chain Antibody Fragments Reveal Potentials for Image-Guided Delivery to Tumors.

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.

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.

Fluorescent liposomes as contrast agents for in vivo optical imaging of edemas in mice.

This study assesses if specially designed fluorescent liposomes can be used as contrast agent for near-infrared fluorescence (NIRF) optical imaging of cultured macrophages in vitro and for NIRF imaging of inflammatory processes, like edema, in an in vivo mouse model. Fluorescent liposomes are prepared by the film hydration and extrusion method using cholesterol, L-phosphatidylcholine, and the NIR fluorescent dye DY-676-C(18) ester. Photon correlation spectroscopy and flow cytometry reveal that fluorescent liposomes are structurally stable for up to 133 days. Distinct uptake/labeling of cultured murine J774 macrophages is demonstrated by confocal laser scanning microscopy (CLSM), flow cytometry, and macroscopic NIRF imaging system at wavelengths >670 nm. Moreover, CLSM analysis reveals fluorescence signals within intracellular compartments. Ear edema is induced in mice (n = 16) by subcutaneous injection of zymosan A. Whole-body NIRF imaging is performed after intravenous injection (0-24 h) of fluorescent liposomes (55 nmol dye per kg body weight). Distinctly higher fluorescence intensities (1613.6 +/- 61.7 a.u.) are detected at inflamed areas of diseased mice as compared to controls (892.8 +/- 19.4 a.u.). Furthermore, cell isolated from ear lavage reveals the presence of labeled F4/80 positive tissue macrophages. Taken together, the results indicate both that mouse macrophages labeled with fluorescent liposomes can be detected in vitro with fluoro-optical methods and that in vivo optical imaging of inflammatory processes with fluorescent liposomes as contrast agent is feasible. Possibly, early stages of other inflammatory diseases could also be detected by the proposed diagnostic tool in the long term.

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".

Single-chain Fv immunoliposomes for the targeting of fibroblast activation protein-expressing tumor stromal cells.

Tumor stromal cells have gained increasing attention as possible target for cancer therapy. Fibroblast activation protein (FAP) represents a cell surface antigen selectively expressed by reactive tumor stromal fibroblasts of various cancers. Here, we describe anti-FAP immunoliposomes as carrier systems for active targeting of FAP-expressing cells. As targeting ligand we used single-chain Fv (scFv) molecules cross-reacting with human and mouse FAP. These scFv molecules were genetically modified to express an additional cysteine residue at the C-terminus allowing a defined and site-directed conjugation. Coupling to Mal-PEG(2000)-DSPE containing liposomes resulted in sterically stabilized scFv immunoliposomes showing strong and specific binding to FAP-expressing cells. These immunoliposomes were highly stable when incubated under physiological conditions (human plasma, 37 degrees C). In addition, we could show that binding to FAP-expressing cells leads to internalization of intact liposomes into the endosomal compartment. Thus, these anti-FAP scFv immunoliposomes should be suitable for target cell-specific delivery and uptake of encapsulated drugs.

Activatable bispecific liposomes bearing fibroblast activation protein directed single chain fragment/Trastuzumab deliver encapsulated cargo into the nuclei of tumor cells and the tumor microenvironment simultaneously.

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. STATEMENT OF SIGNIFICANCE: 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.

In vitro characterization of binding and stability of single-chain Fv Ni-NTA-liposomes.

Recently, we presented a new method for the generation of single-chain Fv (scFv) immunoliposomes, which circumvents the necessity to introduce additional reactive groups in the protein. This method is based on immobilizing scFv fragments via their C-terminal hexahistidyl-tag on liposomes containing nickel-complexed dioleoyl-glycero-succinyl-nitrilotriacetic acid (Ni-NTA-DOGS) as an anchor lipid within the lipid bilayer. Here, we have extended this approach to various other scFv fragments and further demonstrate strong and selective binding of these liposomes to target cells in vitro. In order to evaluate suitability for in vivo applications, we investigated the influence of human plasma on stability and binding behaviour of scFv Ni-NTA-liposomes in vitro using scFv A5 directed against human endoglin (CD105) as a model antibody. We could show that the binding activity to target cells is rapidly lost in the presence of human plasma. Incorporation of polyethylene glycol (PEG) chains into the lipid bilayer did not protect against loss of binding capability. Further studies showed that loss of binding is mainly due to displacement of Ni-NTA-bound scFv fragments caused by plasma proteins. In conclusion, the system allows for a rapid and flexible generation of target cell specific immunoliposomes for in vitro applications but lacks stability for in vivo applications.

Dataset on FAP-induced emergence of spontaneous metastases and on the preparation of activatable FAP-targeting immunoliposomes to detect the metastases.

The underlying data demonstrates that fibroblast activation protein (FAP) paves the way for fibrosarcoma cells, which require the proteolysis of the extracellular matrix (ECM) and basement membranes to intravasate from implanted subcutaneous primary tumors into blood vessels, be transported to distant organs where they extravasate from the blood vessels, reattach and proliferate to metastases. The data additionally shows that FAP, when overexpressed on fibrosarcoma cells induces their invasion and formation of spontaneous metastases in multiple organs, particularly after subcutaneous co-implantation of the FAP-expressing and wildtype fibrosarcoma. The raw and processed data presented herein is related to a research article entitled "Potential of activatable FAP-targeting immunoliposomes in intraoperative imaging of spontaneous metastases" (F.L. Tansi, R. Rüger, C. Böhm, R.E. Kontermann, U.K. Teichgraeber, A. Fahr, I. Hilger, 2016) [1]. Furthermore, evidence for the detection of FAP-expressing tumor cells and cells of the tumor stroma by activatable FAP-targeting liposomes is presented in this dataset.

Potential of activatable FAP-targeting immunoliposomes in intraoperative imaging of spontaneous metastases.

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.

Generation of immunoliposomes using recombinant single-chain Fv fragments bound to Ni-NTA-liposomes.

Recombinant single-chain Fv antibody fragments (scFv) can be combined with liposomes to generate immunoliposomes for targeted drug delivery. Recent studies have shown that scFv molecules modified to express a C-terminal cysteine residue can be used for site-directed chemical conjugation. Here, we present a new method by immobilizing scFv fragments via their C-terminal hexahistidyl-tag on liposomes containing Ni-NTA-lipids (Ni-NTA-DOGS) in their lipid bilayer without the need to introduce additional reactive groups in the protein. Using an anti-endoglin scFv as a model antibody, we could show that scFv molecules are efficiently immobilized on the liposome surface and that these immunoliposomes bind specifically and strongly to endoglin-expressing endothelial cells. This approach allows for a rapid and flexible generation of target cell-specific immunoliposomes.

Bispecific single-chain diabody-immunoliposomes targeting endoglin (CD105) and fibroblast activation protein (FAP) simultaneously.

Liposomes are well-established drug delivery systems with cancer chemotherapy as main focus. To increase the cellular drug delivery, liposomes can be endowed with ligands, e.g. recombinant antibody fragments, which ensure specific cell interaction. Multispecific immunoliposomes can be prepared to improve the liposome to cell interaction by targeting multiple different targets at the same time, for instance by coupling two or more different ligands to the liposomal surface, resulting in a synergistic or additive increase in binding. An alternative approach is the use of bispecific ligands to address at least two different targets. For this purpose we cloned a single-chain diabody fragment (scDb`), a bispecific molecule targeting two antigens, endoglin (CD105) and fibroblast activation protein (FAP), expressed on cells of the tumor microenvironment. As model cell system, a human fibrosarcoma cell line was used expressing endoglin and FAP simultaneously. Monospecific immunoliposomes directed either against endoglin or FAP were compared in vitro for cell binding and cytotoxic activity with bispecific dual-targeted scFv`-IL (bispecific scFv`FAP/CD105-IL) and bispecific single-chain diabody`-IL (scDb`CD105/FAP-IL) targeting endoglin and FAP simultaneously. In the underlying study, bispecific scFv`FAP/CD105-IL interacted stronger with cells expressing FAP and endoglin (both targets simultaneously) compared to the monospecific immunoliposomes. Furthermore, bispecific scDb`-immunoliposomes increased the cell interaction massively and showed enhanced cytotoxicity against target cells using doxorubicin-loaded immunoliposomes. The use of recombinant bispecific ligands as scDb`-molecules facilitates the generation of bispecific immunoliposomes by using the established post-insertion technique, enabling an extension of the ligand specificity spectrum via genetic modification.

Fluorescence-quenching of a liposomal-encapsulated near-infrared fluorophore as a tool for in vivo optical imaging.

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

In vivo near-infrared fluorescence imaging of FAP-expressing tumors with activatable FAP-targeted, single-chain Fv-immunoliposomes.

Molecular and cellular changes that precede the invasive growth of solid tumors include the release of proteolytic enzymes and peptides in the tumor stroma, the recruitment of phagocytic and lymphoid infiltrates and alteration of the extracellular matrix. The reactive tumor stroma consists of a large number of myofibroblasts, characterized by high expression of fibroblast activation protein alpha (FAP). FAP, a type-II transmembrane sialoglycoprotein is an attractive target in diagnosis and therapy of several pathologic disorders especially cancer. In the underlying work, a fluorescence-activatable liposome (fluorescence-quenched during circulation and fluorescence activation upon cellular uptake), bearing specific single-chain Fv fragments directed against FAP (scFv'FAP) was developed, and its potential for use in fluorescence diagnostic imaging of FAP-expressing tumor cells was evaluated by whole body fluorescence imaging. The liposomes termed anti-FAP-IL were prepared via post-insertion of ligand-phospholipid-conjugates into preformed DY-676-COOH-containing liposomes. The anti-FAP-IL revealed a homogeneous size distribution and showed specific interaction and binding with FAP-expressing cells in vitro. The high level of fluorescence quenching of the near-infrared fluorescent dye sequestered in the aqueous interior of the liposomes enables fluorescence imaging exclusively upon uptake and degradation by cells, which results in fluorescence activation. Only FAP-expressing cells were able to take up and activate fluorescence of anti-FAP-IL in vitro. Furthermore, anti-FAP-IL accumulated selectively in FAP-expressing xenograft models in vivo, as demonstrated by blocking experiments using free scFv'FAP. The local tumor fluorescence intensities were in agreement with the intrinsic degree of FAP-expression in different xenograft models. Thus, anti-FAP-IL can serve as a suitable in vivo diagnostic tool for pathological disorders accompanied by high FAP-expression.