Ronny Rüger

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-PEG2000-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°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.

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.

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.

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.

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.

Wheat Germ Agglutinin Modified Liposomes for the Photodynamic Inactivation of Bacteria

Photodynamic inactivation (PDI) of bacteria is a promising approach for combating the increasing emergence of antibiotic resistance in pathogenic bacteria. To further improve the PDI efficiency on bacteria, a bacteria‐targeting liposomal formulation was investigated. A generation II photosensitizer (temoporfin) was incorporated into liposomes, followed by conjugation with a specific lectin (wheat germ agglutinin, WGA) on the liposomal surface. WGA was successfully coupled to temoporfin‐loaded liposomes using an activated phospholipid containing N‐hydroxylsuccinimide residue. Methicillin‐resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa were selected to evaluate the WGA modified liposomes in terms of bacteria targeted delivery and in vitro PDI test. Fluorescence microscopy revealed that temoporfin was delivered to both kinds of bacteria, while flow cytometry demonstrated that WGA‐ modified liposomes delivered more temoporfin to bacteria compared to nonmodified liposomes. Consequently, the WGA‐ modified liposomes eradicated all MRSA and significantly enhanced the PDI of P. aeruginosa. In conclusion, the WGA‐ modified liposomes are a promising formulation for bacteria targeted delivery of temoporfin and for improving the PDI efficiency of temoporfin on both Gram‐positive and Gram‐negative bacterial cells.

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.

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.

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.

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.