Ulrich Massing

Health effects of dietary phospholipids.

Beneficial effects of dietary phospholipids (PLs) have been mentioned since the early 1900s in relation to different illnesses and symptoms, e.g. coronary heart disease, inflammation or cancer. This article gives a summary of the most common therapeutic uses of dietary PLs to provide an overview of their approved and proposed benefits; and to identify further investigational needs.From the majority of the studies it became evident that dietary PLs have a positive impact in several diseases, apparently without severe side effects. Furthermore, they were shown to reduce side effects of some drugs. Both effects can partially be explained by the fact that PL are highly effective in delivering their fatty acid (FA) residues for incorporation into the membranes of cells involved in different diseases, e.g. immune or cancer cells. The altered membrane composition is assumed to have effects on the activity of membrane proteins (e.g. receptors) by affecting the microstructure of membranes and, therefore, the characteristics of the cellular membrane, e.g. of lipid rafts, or by influencing the biosynthesis of FA derived lipid second messengers. However, since the FAs originally bound to the applied PLs are increased in the cellular membrane after their consumption or supplementation, the FA composition of the PL and thus the type of PL is crucial for its effect. Here, we have reviewed the effects of PL from soy, egg yolk, milk and marine sources. Most studies have been performed in vitro or in animals and only limited evidence is available for the benefit of PL supplementation in humans. More research is needed to understand the impact of PL supplementation and confirm its health benefits.

5-Fluorouracil in vesicular phospholipid gels for anticancer treatment: entrapment and release properties

Vesicular phospholipid gels (VPG), i.e. highly concentrated liposomal dispersions, are suitable for entrapping substances such as anticancer drugs with particular high encapsulation efficiencies (EE). We prepared different formulations of VPG with 30% (w/w) lipid containing 5-fluorouracil (5-FU) by high pressure homogenization and analysed their EE and drug release. Using mixtures of hydrogenated soy phosphatidylcholine and cholesterol with molar ratios ranging from 55/45 to 75/25, a decreasing amount of cholesterol correlated with an increasing EE, which is probably due to a reduced amount of smaller vesicles and number of lamellae. Using a 5-FU solution of pH 8.6 for VPG preparation, an EE of approximately 40% was found after redispersion of the gel to a liposomal dispersion and separation of free drug from liposomal drug by size exclusion chromatography. The reduced EE for preparations with lower pH values was attributed to a fast initial drug release due to the increased drug lipophilicity below the pK(a) value of 8. After redispersion of a VPG of pH 8.0, an initially faster release of about a third of the entrapped drug was found during the first 20 min, followed by stable entrapment over many hours. The rapid initial release may be due to the portion of liposomes smaller than 40 nm in diameter, determined by photon correlation spectroscopy. Cryo electron microscopic pictures show a lentil-like shape of these small liposomes. The membrane defects on the edges are probably the reason for the very high initial drug release rate. The half-life time of the release of 5-FU from intact FU-VPG at both pH 7.4 and 8.0 was found to be in the order of 4-5 h and the kinetics are typical for matrix-controlled drug diffusion. The in vitro data of 5-FU loaded VPG suggest their applicability as implants with controlled release properties or, after redispersion, as intravenously injected liposomal formulations.

Biophysical and lipofection studies of DOTAP analogs.

In order to investigate the relationship between lipid structure and liposome-mediated gene transfer, we have studied biophysical parameters and transfection properties of monocationic DOTAP analogs, systematically modified in their non-polar hydrocarbon chains. Stability, size and (by means of anisotropy profiles) membrane fluidity of liposomes and lipoplexes were determined, and lipofection efficiency was tested in a luciferase reporter gene assay. DOTAP analogs were used as single components or combined with a helper lipid, either DOPE or cholesterol. Stability of liposomes was a precondition for formation of temporarily stable lipoplexes. Addition of DOPE or cholesterol improved liposome and lipoplex stability. Transfection efficiencies of lipoplexes based on pure DOTAP analogs could be correlated with stability data and membrane fluidity at transfection temperature. Inclusion of DOPE led to rather uniform transfection and anisotropy profiles, corresponding to lipoplex stability. Cholesterol-containing lipoplexes were generally stable, showing high transfection efficiency at low relative fluidity. Our results demonstrate that the efficiency of gene transfer mediated by monocationic lipids is greatly influenced by lipoplex biophysics due to lipid composition. The measurement of fluorescence anisotropy is an appropriate method to characterize membrane fluidity within a defined system of liposomes or lipoplexes and may be helpful to elucidate structure-activity relationships.

Plasma lyso-phosphatidylcholine concentration is decreased in cancer patients with weight loss and activated inflammatory status

BackgroundIt has been observed that ras-transformed cell lines in culture have a higher phosphatidylcholine (PC) biosynthesis rate as well as higher PC-degradation rate (increased PC-turnover) than normal cells. In correspondence to these findings, the concentrations of the PC-degradation product lyso-phosphatidylcholine (LPC) in cancer patients were found to be decreased. Our objective was the systematic investigation of the relationship between LPC and inflammatory and nutritional parameters in cancer patients. Therefore, plasma LPC concentrations were assessed in 59 cancer patients and related to nutritional and inflammatory parameters. To determine LPC in blood plasma we developed and validated a HPTLC method.ResultsAverage plasma LPC concentration was 207 ± 59 μM which corresponds to the lower limit of the reported range in healthy subjects. No correlation between LPC and age, performance status, body mass index (BMI) or fat mass could be seen. However, LPC correlated inversely with plasma C-reactive protein (CRP) and whole blood hydrogen peroxides (HPO). Further, a negative correlation could be observed between LPC and whole body extra cellular fluid volume (ECF) as well as with relative change in body weight since cancer diagnosis.ConclusionIn conclusion, LPC concentrations were decreased in cancer patients. LPC plasma concentrations correlated with weight loss and inflammatory parameters and, therefore, might be a general indicator of severity of malignant disease.

Surveillance of siRNA integrity by FRET imaging

Techniques for investigation of exogenous small interfering RNA (siRNA) after penetration of the cell are of substantial interest to the development of efficient transfection methods as well as to potential medical formulations of siRNA. A FRET-based visualization method including the commonplace dye labels fluorescein and tetramethylrhodamin (TMR) on opposing strands of siRNA was found compatible with RNA interference (RNAi). Investigation of spectral properties of three labelled siRNAs with differential FRET efficiencies in the cuvette, including pH dependence and FRET efficiency in lipophilic environments, identified the ratio of red and green fluorescence (R/G-ratio) as a sensitive parameter, which reliably identifies samples containing >90% un-degraded siRNA. Spectral imaging of siRNAs microinjected into cells showed emission spectra indistinguishable from those measured in the cuvette. These were used to establish a calibration curve for assessing the degradation state of siRNA in volume elements inside cells. An algorithm, applied to fluorescence images recorded in standard green and red fluorescence channels, produces R/G-ratio images of high spatial resolution, identifying volume elements in the cell with high populations of intact siRNA with high fidelity. To demonstrate the usefulness of this technique, the movement of intact siRNA molecules are observed after introduction into the cytosol by microinjection, standard transfection and lipofection with liposomes.

Antimetastatic Effects of Liposomal Gemcitabine and Empty Liposomes in an Orthotopic Mouse Model of Pancreatic Cancer

Objectives: Test the efficacy of liposomal gemcitabine (GemLip) on primary tumor and metastases using the pancreatic tumor cell line AsPC1 implanted orthotopically into nude mice. Methods: The efficacy of gemcitabine and GemLip cells was tested on luciferase-transduced AsPC1 cells in vitro as well as implanted orthotopically into the pancreata of nude mice. Results: In vitro, the IC50s for GemLip and gemcitabine were 20 nM and 140 nM, respectively. However, when applied against established tumors, GemLip (8 mg/kg) blocked tumor growth for 5 consecutive weeks according to bioluminescence measurements in vivo. Gemcitabine (240 mg/kg) had no effect on luciferase-monitored tumor growth. When analyzed at the time of necropsy, GemLip strongly reduced tumor size (−64% ± [SD] 27%; ***P < 0.0001), whereas gemcitabine only weakly (−36% ± 37%) affected tumor size. Empty liposomes had no effect on the tumor size. GemLip and empty liposomes both significantly interfered with the metastatic spread to the liver, as measured using luciferase assays (GemLip, *P = 0.01; empty liposomes, *P = 0.036). In addition, they showed effects against spleen, as well as peritoneal metastases. Conclusions: GemLip presents an effective new formulation of gemcitabine, combining the targeting and protective features of the liposomes with their antimetastatic effects to target pancreatic cancer.

Quantification of various phosphatidylcholines in liposomes by enzymatic assay.

The purpose of this research was to adapt a colorimetric, phospholipase D-based serum-phospholipid assay for the quantification of phosphatidylcholine (PC) in liposomes using a microtitre plate reader. PC from natural egg PC liposomes was quantified reliably. In contrast, poor sensitivity was found for liposomes composed of saturated PCs (dipalmitoyl-phosphatidylcholine [DPPC], hydrogenated egg PC). Triton X-100 was then added to the liposomes followed by heating above the phase transition temperature. This modified sample preparation resulted in recoveries of 102.6%±1.0%, 104.4%±7.6%, and 109.4%±3.2% for E80, E80-3/cholesterol, and DPPC liposomes, respectively. Absolute quantification of unknown PCs against a choline chloride standard is feasible, but relative measurements against the very same PC are recommended wheneve possible. Validation experiments revealed an absolute quantification limit of 1.25 μg per assay, a good linearity in the range of 25 to 1000μg/mL PC (r2≥0.9990) and a quite high accuracy (99.8%–101.4% of theory) and precision (relative standard deviation ≤3.2%) for all 3 PCs studied. The method is thus regarded as suitable for sensitive, rapid, and reliable routine quantification of PCs in liposomes.

Uptake of apolipoprotein E fragment coupled liposomes by cultured brain microvessel endothelial cells and intact brain capillaries

The suitability of surface modified liposomes as drug carriers for brain-specific targeting was investigated using apolipoprotein E fragments as brain-directed vectors. Liposomes coated with polyethylene glycol-2000 (sterically stabilized, PEGylated liposomes) were prepared from hydrogenated egg phosphatidylcholine, cholesterol, and a PEG-derivatized phospholipid. Liposomes were covalently coupled to a peptide of 26 amino acids length, derived from the binding site of human apolipoprotein E4 (ApoE4) and a peptide of random amino acid sequence, respectively. Rhodamine-labeled dipalmitoylphosphatidylethanolamine was incorporated into the lipid bilayer in order to visualize the liposomal interaction with brain capillary endothelial cell monolayers. The interaction of the liposomes with monolayers of porcine brain capillary endothelial cells (BCEC), the rodent cell line RBE4, and freshly isolated porcine brain capillaries was studied by means of confocal laser scanning fluorescence microscopy. In contrast to random peptide coupled liposomes, the ApoE4-fragment coupled liposomes were rapidly taken up by cultured BCECs and RBE4 cells. Uptake could be inhibited by ApoE4, free peptide, and antibodies against the LDL receptor in a concentration-dependent manner. The results indicate that the liposomes are internalized via the LDL receptor, which is expressed at the blood−brain barrier. In conclusion, liposomes coupled to ApoE4 fragments are taken up into brain endothelium via an endocytotic pathway and may therefore be a suitable carrier for drug delivery to the brain.

Lysophosphatidylcholine Pretreatment Reduces VLA-4 and P-Selectin–Mediated B16.F10 Melanoma Cell Adhesion In vitro and Inhibits Metastasis-Like Lung Invasion In vivo

Lysophosphatidylcholine (LysoPC) is an important intermediate in degradation and biosynthesis of phosphatidylcholine (PC). Reduced plasma LysoPC levels observed in patients with advanced cancer indicate a deregulation of LysoPC metabolism in metastasis. Recent data showed strong antimetastatic effects of liposomes consisting of saturated PC in a murine pancreatic metastasis model. LysoPC, generated from saturated PC after accumulation of the liposomes in tumor tissue, might be contributing to these effects. Examining effects of high local concentrations of saturated LysoPC and investigating potential molecular mechanisms, fast removal of saturated LysoPC from medium by murine B16.F10 melanoma cells and radical shifts in tumor cell membrane fatty acid (FA) composition toward saturated FAs were observed in vitro. Scanning electron microscopy revealed remarkable morphologic surface changes of LysoPC-treated tumor cells, probably causing their impaired migratory ability on fibronectin. A LysoPC concentration exceeding a threshold of about 400 μmol/L, slightly above physiologic levels, strongly reduced VLA-4–mediated binding of B16.F10 cells to VCAM-1 as well as P-selectin–dependent interaction with activated platelets, although expression levels were not altered. These findings were reflected in a syngenic intravenous lung invasion model using repeatedly ex vivo LysoPC-treated (450 μmol/L) B16.F10 cells, resulting in significantly reduced lung metastasis-like lesions (−48.3%, P = 0.006). Prior application of 50 IU unfractionated heparin further reduced lung invasion (−81.6%, P = 0.043). Our work shows for the first time that saturated LysoPC in high concentrations reduces melanoma cell adhesion in vitro and hematogeneous dissemination in vivo by direct ex vivo tumor cell targeting. Mol Cancer Ther; 10(1); 186–97. ©2011 AACR.

Killing tumour cells by alkylphosphocholines: evidence for involvement of CD95.

Many lipids act as cellular messengers and lead to a variety of different cellular responses. Out of the group of these compounds the ceramides are able to induce apoptosis, and some synthetic lipids can mimic this effect. Apoptosis is an important mechanism whereby chemotherapeutics exhibit their anti-oncogenic activity. Although, some lipid analogues were used in clinical trials, they exert severe side effects and their mechanism of action is widely unknown. We present here a new class of synthetic alkylphosphocholines (APC) that induce programmed cell death in leukaemia cells. The signs of apoptosis arise after 1 h of incubation with these compounds as shown by phosphatidylserine externalisation followed by caspase activation and DNA fragmentation. We demonstrate that the molecular target of these lipids is upstream of caspases and Bcl-2. Experiments with FADD dominant negative cells reveal that induction of apoptosis occurs on the level of CD95 and that these compounds can now be optimised for their capacity to activate the apoptosis-inducing receptor CD95.