Biodistribution of the photosensitizer temoporfin after in vivo topical application of temoporfin-loaded invasomes in mice bearing subcutaneously implanted HT29 tumor.

Temoporfin (mTHPC) has a great potential for the topical photodynamic therapy. However, it presents a highly hydrophobic second generation photosensitizer with low percutaneous penetration. In order to use mTHPC for dermal/transdermal delivery it is necessary to employ some of the penetration enhancement methods. In this study invasomes were used as a highly effective drug nanocarrier system to enhance its skin penetration, being composed of non-hydrogenated soybean lecithin (10% w/v), ethanol (3.3%w/v), a mixture of terpenes (1% w/v of the mixture cineole:citral:d-limonene = 45:45:10 v/v) and phosphate buffer saline up to 100% w/v. A pharmacokinetic/biodistribution study was performed in mice bearing s.c. implanted human colorectal tumor HT29 upon the application of mTHPC-loaded invasomes onto the skin above the underlying tumor. The aim was to obtain the biodistribution profile of mTHPC i.e. to gain data on mTHPC-distribution in the body (tumor, treated skin, muscle, blood, liver and untreated skin) of mice after the topical application of mTHPC-loaded invasomes. The results revealed that a significant mTHPC-amount was found in treated skin already after 2 h of incubation time. As to the tumor, significant amounts were found after 12 h, while the highest mTHPC-amount was found after 24 h. This study showed that invasomes applied onto the skin may deliver mTHPC to the tumor being necessary for PDT. Since mTHPC was also found in blood and liver, transdermal mTHPC delivery was confirmed. In conclusion, mTHPC-invasomes could be used for topical PDT of cutaneous and subcutaneous lesions, however with general photoxicity induced by systemic apsorption of mTHPC lasting only for 2 weeks. Additionally, due to systemic absorption of mTHPC after invasomes application onto the skin, they could be used transdermally for the PDT treatment of diseases, which need systemic drug absorption. However, it should be emphasized that mice were used in the study, differing in the skin properties compared to human skin. Thus, additional studies should be conducted.

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.

Development of hydrophilic gels containing coenzyme Q10-loaded liposomes: characterization, stability and rheology measurements.

OBJECTIVE: The aim of this study was to develop, characterize and evaluate stability of a gel containing coenzyme Q10 (Q10)-loaded liposomes, and enhance the stability of Q10 in the nanocarrier-containing gel compared to the conventional gel. METHODS: Q10-loaded liposome dispersions prepared from unsaturated or saturated lecithin, were characterized for particle size, polydispersity index (PDI), zeta-potential, pH value, oxidation index, Q10-content and morphology, and incorporated into carbomer gel. Liposome gels and liposome-free gel were analyzed for flow properties, pH values, Q10-content, and liposomes size and PDI (liposome gels), 48 h after preparation and in predetermined time intervals during 6 months storage at different temperatures in order to predict their long term stability. RESULTS: Liposomes were of small particle size, homogeneous, negatively charged, and their incorporation into gel did not significantly change (p > .05) their particle size and PDI. All gels revealed non-Newtonian, shear-thinning plastic flow behavior during storage with no marked changes in rheological parameters. Storage of gels did not significantly influence the pH value (p > .05), while it significantly decreased Q10-content (p < .05). Q10 was significantly more (p < .05) stable in liposome gel containing unsaturated lecithin liposomes (G1) than in gel containing saturated lecithin liposomes (G2) and liposome-free gel (G3). CONCLUSIONS: Q10-loaded liposome gel G1 was the optimal formulation, since during storage at different temperatures, it did not show significant increase in liposome size and PDI, it provided significantly higher stability for Q10 than other gels and its pH value was suitable for skin application. Due to limited Q10-stability it should be stored at 4 °C.

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".

Lipid Architectonics for Superior Oral Bioavailability of Nelfinavir Mesylate: Comparative in vitro and in vivo Assessment.

Nelfinavir mesylate (NFV), a human immunodeficiency virus (HIV) protease inhibitor, is an integral component of highly active anti retro viral therapy (HAART) for management of AIDS. NFV possesses pH-dependent solubility and has low and variable bioavailability hampering its use in therapeutics. Lipid-based particulates have shown to improve solubility of poorly water soluble drugs and oral absorption, thereby aiding in improved bioavailability. The current study compares potential of vesicular and solid lipid nanocarriers of NFV with drug nanocrystallites and microvesicular systems like cochleates in improving bioavailability of NFV. The paper outlines investigation of systems using in vitro models like in vitro lipolysis, in vitro release, and permeation through cell lines to predict the in vivo potential of nanocarriers. Finally, in vivo pharmacokinetic study is reported which provided proof of concept in sync with results from in vitro studies. Graphical Abstract ᅟ.

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.

Investigations of the influence of liposome composition on vesicle stability and drug transfer in human plasma: a transfer study.

Liposomal delivery constitutes a promising approach for i.v. administration of temoporfin (mTHPC) because lipid membranes can host these drug molecules. This study investigates the transfer and release of mTHPC to plasma proteins and stability of various liposomal formulations. To this end, we employed traces of radioactive markers and studied the effects of fatty acid chain length and the degree of saturation in the lipophilic tail, addition of cholesterol and PEGylation of the membrane surface and different drug-to-lipid ratios (DLRs). Liposomes were incubated in human plasma for various incubation times. Drawn samples were separated by asymmetrical flow field-flow fractionation (AF4). Drug was recovered in four fractions identified as albumin, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and liposomes. Our results suggest that mTHPC fits best into fluid, unmodified bilayers when the drug-to-lipid ratio is low. Membrane rigidification as well as the presence of cholesterol and PEGyated lipids reduced the ability of the membrane to accommodate the drug but simultaneously improved the vesicle stability in plasma. Both mechanisms jointly affect the total degree of mTHPC release. We analyzed our data using a kinetic model that suggests the drug to be associated with the host membrane in two distinct states of which only one interacts directly with the plasma compartment.

Tip-enhanced Raman scattering for tracking of invasomes in the stratum corneum.

Stratum corneum is the primary skin barrier to percutaneous absorption. Since 1980, topical liposomal formulations have been proposed and successfully employed for increasing the drug penetration through the skin. There is no clear consensus on the drug penetration mechanism from topically applied liposomes, despite a vast amount of research. One of the reasons for the ambiguity is that the interactions between the stratum corneum and liposomes are in nanoscale, which makes them difficult to probe. In this study, we employed tip-enhanced Raman scattering (TERS) to gain a better understanding of the interactions between the human stratum corneum and topically applied liposomal system called invasomes. TERS is capable of imaging at nanometer spatial resolution and can provide structural information at the nanometer scale. A sample preparation technique was developed and calibrated to enable TERS on complex stratum corneum samples. Invasomes prepared from a head deuterated phospholipid were employed to aid identification of topically applied liposomal phospholipid in the stratum corneum. Results presented in this study give for the first time a strong spectroscopic evidence along with high-resolution images to show intact invasome vesicles deep in the stratum corneum upon topical application.

Vesicle aggregates as a model for primitive cellular assemblies.

Primitive cell models help to understand the role that compartmentalization plays in origin of life scenarios. Here we present a combined experimental and modeling approach towards the construction of simple model systems for primitive cellular assemblies. Charged lipid vesicles aggregate in the presence of oppositely charged biopolymers, such as nucleic acids or polypeptides. Based on zeta potential measurements, dynamic light scattering and cryo-transmission electron-microscopy, we have characterized the behavior of empty and ferritin-filled large unilamellar POPC vesicles, doped with different amounts of cationic (DDAB, CTAB) and anionic (sodium oleate) surfactants, and their aggregation upon the addition of anionic (tRNA, poly-l-glutamic acid) and cationic (poly-l-arginine) biopolymers, respectively. The experimental results are rationalized by a phenomenological modeling approach that predicts the average size of the vesicle aggregates as function of the amount of added biopolymers. In addition, we discuss the mechanism of vesicle aggregation induced by oppositely charged biopolymers. Our study complements previous reports about the formation of giant vesicle clusters and thus provides a general vista on primitive cell systems, based on the association of vesicles into compartmentalized aggregates.

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.

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.

Micro-spherical cochleate composites: method development for monodispersed cochleate system.

Cochleates have been of increasing interest in pharmaceutical research due to their extraordinary stability. However the existing techniques used in the production of cochleates still need significant improvements to achieve sufficiently monodispersed formulations. In this study, we report a simple method for the production of spherical composite microparticles (3-5 µm in diameter) made up of nanocochleates from phosphatidylserine and calcium (as binding agent). Formulations obtained from the proposed method were evaluated using electron microscopy and small angle X-ray scattering and were compared with conventional cochleate preparation techniques. In this new method, an ethanolic lipid solution and aqueous solution of a binding agent is subjected to rapid and uniform mixing with a microfluidic device. The presence of high concentration of organic solvent promotes the formation of composite microparticles made of nanocochleates. This simple methodology eliminates elaborate preparation methods, while providing a monodisperse cochleate system with analogous quality.

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.

Transdermal delivery from liposomal formulations - Evolution of the technology over the last three decades.

Strong barrier properties of stratum corneum often limits the efficiency of drug delivery through skin. Several strategies were tried to improve permeation of drug through skin for local as well as systemic drug delivery. Incorporation of the drug within flexible liposomal vesicles has been one of the popular and well-studied approaches for delivering drug to deeper layers of the skin or even systemic circulation. Flexible/deformable/elastic liposomal systems such as invasomes, Transfersomes®, ethosomes, niosomes, etc. have demonstrated encouraging results in delivering small molecules and large proteins to the skin. It is necessary to recognize the promising concepts and analyze their potential, since a clear understanding of the drawbacks and advantages of these approaches will lead towards future development. In the current review we have attempted to give an overview of different liposomal drug carriers for transdermal drug delivery and their efficiency as drug delivery system through different in vivo and in vitro studies. Also, an overview of the studies which investigated the interactions between skin and vesicles, which have lead us to our current understanding of the skin penetration mechanisms of liposomal formulations is presented.

Lecithin and PLGA-based self-assembled nanocomposite, Lecithmer: preparation, characterization, and pharmacokinetic/pharmacodynamic evaluation.

The present study investigates the drug delivery potential of polymer lipid hybrid nanocomposites (Lecithmer®) composed of poly(D,L-lactide-co-glycolide (PLGA) and soya lecithin. Core-shell structure of Lecithmer was evident from cryo-TEM images. Daunorubicin (DNR) and lornoxicam (LNX)-incorporated Lecithmer nanocomposites were evaluated for anticancer and anti-inflammatory activity. DNR- and LNX-loaded Lecithmer had mean particle size of ∼335 and ∼282.7 nm, respectively. Lecithmer formulated with different cationic lipids resulted in lower particle size (∼120 nm) and positive zeta potential. Entrapment efficiency of DNR and LNX was 93.16 and 88.59 %, respectively. In vitro release of DNR from Lecithmer was slower compared to PLGA nanoparticles. DNR release from Lecithmer was significantly higher at pH 5.5 (80.96 %) as compared to pH 7.4 (55.95 %), providing advantage for selective tumor therapy. Similarly, sustained release of LNX (30 % in 10 h) was observed at pH 7.4. DNR in Lecithmer showed superior cytotoxicity on human erythroleukemic K562 cells. Pharmacokinetic study in Wistar rats with i.v. administered DNR-loaded Lecithmer showed higher volume of distribution, lower elimination rate constant, and longer half-life (81.68 L, 0.3535 h(-1), 1.96 h) as compared to DNR solution (57.46 L, 0.4237 h(-1), 1.635 h). Pharmacodynamic evaluation of orally administered LNX-loaded Lecithmer showed superior anti-inflammatory activity with maximum inhibition of 81.2 % vis-à-vis 53.57 % in case of LNX suspension. In light of these results, Lecithmer can be envisaged as a promising nanosystem for parenteral as well as oral drug delivery.

Design of cholesterol arabinogalactan anchored liposomes for asialoglycoprotein receptor mediated targeting to hepatocellular carcinoma: In silico modeling, in vitro and in vivo evaluation.

We have developed active targeting liposomes to deliver anticancer agents to ASGPR which will contribute to effective treatment of hepatocellular carcinoma. Active targeting is achieved through polymeric ligands on the liposome surface. The liposomes were prepared using reverse phase evaporation method and doxorubicin hydrocholoride, a model drug, was loaded using the ammonium sulphate gradient method. Liposomes loaded with DOX were found to have a particle size of 200nm with more than 90% entrapment efficiency. Systems were observed to release the drug in a sustained manner in acidic pH in vitro. Liposomes containing targeting ligands possessed greater and selective toxicity to ASGPR positive HepG2 cell lines due to specific ligand receptor interaction. Bio-distribution studies revealed that liposomes were concentrated in the liver even after 3h of administration, thus providing conclusive evidence of targeting potential for formulated nanosystems. Tumor regression studies indicated greater tumor suppression with targeted liposomes thereby establishing superiority of the liposomal system. In this work, we used a novel methodology to guide the determination of the optimal composition of the targeting liposomes: molecular dynamics (MD) simulation that aided our understanding of the behaviour of the ligand within the bilayer. This can be seen as a demonstration of the utility of this methodology as a rational design tool for active targeting liposome formulation.

Understanding cochleate formation: insights into structural development.

Understanding the structure and the self-assembly process of cochleates has become increasingly necessary considering the advances of this drug delivery system towards the pharmaceutical industry. It is well known that the addition of cations like calcium to a dispersion of anionic lipids such as phosphatidylserines results in stable, multilamellar cochleates through a spontaneous assembly. In the current investigation we have studied the intermediate structures generated during this self-assembly of cochleates. To achieve this, we have varied the process temperature for altering the rate of cochleate formation. Our findings from electron microscopy studies showed the formation of ribbonlike structures, which with proceeding interaction associate to form lipid stacks, networks and eventually cochleates. We also observed that the variation in lipid acyl chains did not make a remarkable difference to the type of structure evolved during the formation of cochleates. More generally, our observations provide a new insight into the self-assembly process of cochleates based on which we have proposed a pathway for cochleate formation from phosphatidylserine and calcium. This knowledge could be employed in using cochleates for a variety of possible biomedical applications in the future.

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.

Quantitative In Vitro Assessment of Liposome Stability and Drug Transfer Employing Asymmetrical Flow Field-Flow Fractionation (AF4).

PURPOSE: In the present study we introduce an efficient approach for a size-based separation of liposomes from plasma proteins employing AF4. We investigated vesicle stability and release behavior of the strongly lipophilic drug temoporfin from liposomes in human plasma for various incubation times at 37°C. METHODS: We used the radioactive tracer cholesteryl oleyl ether (COE) or dipalmitoyl-phosphocholine (DPPC) as lipid markers and (14)C-labeled temoporfin. First, both lipid labels were examined for their suitability as liposome markers. Furthermore, the influence of plasma origin on liposome stability and drug transfer was investigated. The effect of membrane fluidity and PEGylation on vesicle stability and drug release characteristics was also analyzed. RESULTS: Surprisingly, we observed an enzymatic transfer of (3)H-COE to lipoproteins due to the cholesterol ester transfer protein (CETP) in human plasma in dependence on membrane rigidity and were able to inhibit this transfer by plasma preincubation with the CETP inhibitor torcetrapib. This effect was not seen when liposomes were incubated in rat plasma. DPPC labels suffered from hydrolysis effects during preparation and/or storage. Fluid liposomes were less stable in human plasma than their PEGylated analogues or a rigid formulation. In contrast, the transfer of the incorporated drug to lipoproteins was higher for the rigid formulations. CONCLUSIONS: The observed effects render COE-labels questionable for in vivo studies using CEPT-rich species. Here, choline labelled (14)C-DPPC was found to be the most promising alternative. Bilayer composition has a high influence on stability and drug release of a liposomal formulation in human plasma.