Supramolecular assemblies of a new class of nonplanar cationic metalloporphyrins and anionic metallophthalocyanines

The heteroaggregation behavior between a new class of nonplanar cationic β-octabrominated meso-alkylpyridinium zinc(II)-porphyrins (β-Br₈(ZnP)) and anionic tetrasulfonated metallophthalocyanines (MTSPc, M = Ni(II) and Cu(II)) has been studied by UV-Vis electronic spectroscopy, in dimethylsulfoxide (DMSO) solution. The heteroaggregate stoichiometry and the association constants were determined by means of Job plots. Dimers and unexpected trimers, taking into account the existence of axially coordinated DMSO molecules to the central metal in both β-Br₈(ZnP) and MTSPc complexes, are formed in solution. The spectroscopic properties of the heteroaggregates are markedly different from those observed in the correspondent planar cationic derivatives, the heteroaggregates showing major changes predominantly in the β-Br₈(ZnP) Soret band region and minor effects in the MTSPc Q bands. The observed changes in the Soret band region (red/blue shifts, decrease in the absorption intensities) depend on the nature of the alkyl substituent attached to the meso-pyridinium group. The greater versatility of the nonplanar porphyrins accommodating the meso-substituents in out-of-plane and in-plane conformations is proposed to explain the observed stoichiometries and the differences on the heteroaggregates spectroscopic properties for each β-Br₈ZnP compound. The likely conformations assumed by the meso-substituents in these β-Br₈(ZnP) compounds and its spectroscopic characteristics are in accordance with the participation of the substituents as the main factor on the extent of the observed red-shifted spectra in nonplanar porphyrins. The obtained association constants (K_IP) for the dimers and trimers are lower than those previously found for the similar planar cationic porphyrin systems, due to the lack of extensive π-π interactions and to the less effective approximation between the ionic groups, resulting in loosened heteroaggregates, particularly for the trimeric systems. Furthermore, the experimental results suggest that the NiTSPc is more distorted in DMSO solution than the CuTSPc derivative, favoring the interaction with the nonplanar β-Br₈(ZnP) compounds.     

Synergistic Effect of Phospholipid-Based Liposomes and Propylthiouracil on U-937 Cell Growth

Scientific evidence indicates that exogenous phospholipids in the form of liposomes can affect cell growth. Effects of liposomes on cell growth depend on several factors including composition of liposomes, lipid concentration, and type of cells studied. Because phagocytic cells such as monocytes and macrophages are natural targets of liposomes, intracellular delivery of drugs to modulate cellular activity of these cells is of interest. We explored the effects of phospholipid-based liposomes composed of soy bean phosphatidylcholine (PC) as the main lipid component on U-937 cell growth. Effects of charge-imposing lipids and cholesterol were also studied. In addition, we investigated whether phospholipid-based liposomes would exert any interaction on cell growth with propylthiouracil, a drug with known antiproliferative activity. We found that PC in the form of extruded liposomes had intrinsic antiproliferative activity on U‐937 cells at concentrations of 200 μM and up without any appreciable cytotoxiciy. Phosphatidylserine and phosphtidylglycerol, but not dicetlylphosphate, at 10 mol% increased growth retardation activity of PC liposomes. Cholesterol at 30 mol% did not have any effect on cell growth, except for liposomes composed of PC and phosphatidylserine, where growth retardation was negated in the presence of cholesterol. Synergistic effect on cell growth was seen with certain liposome compositions when 5.5 μg/mL of propylthiouracil was coincubated. The results of this study suggest that the effects of exogenous lipids on cell growth should be taken into consideration when PC-based liposomes are to be used as drug delivery systems, especially when the targets are cells with phagocytic activity.     

A nanosensory device fabricated on a liposome for detection of chemical signals

This paper describes construction of a nanosensory device for amplified detection of biologically important amines as chemical signals. The device was inspired by a biological signal transduction system, and was fabricated on an artificial cell membrane through self‐organization of the molecular components, such as a synthetic receptor and a natural enzyme. Selective recognition of biologically important amines was achieved by a synthetic receptor with a pyridoxal moiety, as evaluated by means of electronic absorption spectroscopy. The selectivity in detecting amines as chemical signals mainly depends on hydrophobicity of the amines. The event upon detecting the chemical signals was transmitted to an enzyme by a metal ion acting as a mediator species, and then the enzyme amplified the event by the catalytic reaction to obtain signal output. This paper is realization of a biomimetic signal transduction system using amines as chemical signals and may provide a useful guidepost for designing integrated nanosystems. Biotechnol. Bioeng. 2010;105: 37–43. © 2009 Wiley Periodicals, Inc.     

The development and application of protein-liposome conjugation techniques

Numerous techniques have been developed over the past 10 years for the conjugation of proteins to liposomes. Early procedures involved coupling with reagents such as glutaraldehyde or EDCI. Subsequently, more sophisticated approaches involving selective bifunctional coupling agents have been developed. These later procedures are also much more efficient for coupling in aqueous media. The techniques of coupling have become more rigorous because investigators have recognized the inherent problems of producing, purifying and characterizing protein conjugated liposomes.

Protein-liposome coupling techniques were developed mainly for targeted drug delivery. The attachment of specific antibodies to the surface of the liposomes makes them able to bind to cells and to subsequently be internalised by the cells. Protein conjugated liposomes have also been used for various immunochemical and diagnostic purposes. These include the binding of labelled liposomes to cells and the agglutination of cells or latex particles by protein conjugated liposomes.     

Interactions between model membranes and lignin-related compounds studied by immobilized liposome chromatography

In order to elucidate the modes of interaction between lignin precursors and membranes, we have studied the influence of temperature, lipid composition and buffer composition on the partitioning of monolignol and dilignol model substances into phospholipid bilayers. The partitioning was determined by immobilized liposome chromatography, which is an established method for studies of pharmaceutical drugs but a new approach in studies of lignin synthesis. The temperature dependence of the retention and the effect of a high ammonium sulfate concentration in the mobile phase demonstrated that the interaction involved both hydrophobic effects and polar interactions. There was also a good correlation between the partitioning and the estimated hydrophobicity, in terms of octanol/water partitioning. The partitioning behavior of the model substances suggests that passive diffusion over the cell membrane is a possible transport route for lignin precursors. This conclusion is strengthened by comparison of the present results with the partitioning of pharmaceutical drugs that are known to pass cell membranes by diffusion.     

Temperature-dependent permeability of large unilamellar liposomes

The temperature-dependent drug leakage from liposomes composed of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (4:1, by weight) was studied. Experiments were performed in Hepes buffer and 50% fetal calf serum. Large unilamellar liposomes were formed by the reverse phase evaporation process and extruded through a series of polycarbonate membranes with pore sizes of 0.4, 0.2, 0.1 and 0.08 μm. The release of the water soluble radioisotopes cytosine 1-β-D-[³H]arabinofuranoside and [³H]inulin from the aqueous compartment of these liposomes was measured as a function of time and temperature. Both radioisotopes were released at temperatures near 42°C, the solid-to-liquid-crystalline phase transition temperature of these lipids. The percent drug release decreased as the size of the liposomes was reduced. This effect was more pronounced in Hepes buffer than serum. The release of both radioisotopes was greatest at 40°C in Hepes buffer and at 43°C in 50% fetal calf serum. In addition, the rate of drug release was much faster in serum than in buffer. These results suggest that different drug release processes are occurring in buffer and in serum.     

Functional Reconstitution of a Prokaryotic K+ Channel

SliK, a K⁺ channel encoded by the Streptomyces KcsA gene, was expressed, purified, and reconstituted in liposomes. A concentrative ⁸⁶Rb⁺ flux assay was used to assess the ion transport properties of SliK. SliK-mediated ionic flux shows strong selectivity for K⁺ over Na⁺ and is inhibited by micromolar concentrations of Ba²⁺, mirroring the basic permeation characteristic of eukaryotic K⁺ channels studied by electrophysiological methods. ⁸⁶Rb⁺ uptake kinetics and equilibrium measurements also demonstrate that the purified protein is fully active.     

Association of vasoactive intestinal peptide with polymer-grafted liposomes: Structural aspects for pulmonary delivery

A polymer-grafted liposomal formulation that has the potential to be developed for aerosolic pulmonary delivery of vasoactive intestinal peptide (VIP), a potent vasodilatory neuropeptide, is described. As VIP is prone to rapid proteolytic degradation in the microenvironment of the lung a proper delivery system is required to increase the half-life and bioavailability of the peptide. Here we investigate structural parameters of unilamellar liposomes composed of palmitoyl-oleoyl-phosphatidylcholine, lyso-stearyl-phosphatidylglycerol and distearyl-phosphatidyl-ethanolamine covalently linked to polyethylene glycol 2000, and report on VIP-lipid interaction mechanisms. We found that the cationic VIP is efficiently entrapped by the negatively charged spherical liposomes and becomes converted to an amphipathic α-helix. By fluorescence spectroscopy using single Trp-modified VIP we could show that VIP is closely associated to the membrane. Our data suggest that the N-terminal random-coiled domain is embedded in the interfacial headgroup region of the phospholipid bilayer. By doing so, neither the bilayer thickness of the lipid membrane nor the mobility of the phospholipid acyl chains are affected as shown by small angle X-ray scattering and electron spin resonance spectroscopy. Finally, in an ex vivo lung arterial model system we found that liposomal-associated VIP is recognized by its receptors to induce vasodilatory effects with comparable high relaxation efficiency as free VIP but with a significantly retarded dilatation kinetics. In conclusion, we have designed and characterized a liposomal formulation that is qualified to entrap biologically active VIP and displays structural features to be considered for delivery of VIP to the lung.     

Physiological role of phosphatidylcholine in the Pseudomonas putida A ATCC 12633 response to tetradecyltrimethylammonium bromide and aluminium

Aims:  To evaluate the effect of tetradecyltrimethylammonium bromide (TTAB) and aluminium stresses on the phospholipid (PL) composition of Pseudomonas putida A ATCC 12633.
Methods and Results:  Pseudomonas putida were grown with TTAB in the presence or absence of AlCl₃, and the PL composition was analysed. The presence of TTAB resulted in an increase in phosphatidylglycerol and phosphatidic acid levels (6- and 20-fold, respectively) with respect to the levels in cells grown without the surfactant. With AlCl₃, phosphatidylcholine (PC) increased (threefold) and cell-free extracts contained approximately threefold more phosphatidylcholine synthase activities than extracts without AlCl₃, indicating that the PC level is dependent upon activation of this enzyme.
Conclusions:  The negative charges of the headgroups of PL are the primary membrane-associated factors for the response to TTAB. PC are involved in cellular responses to binding Al³⁺ and should be viewed as a temporary reservoir of available Al³⁺ to allow a more efficient utilization of TTAB by Ps. putida .
Significance and Impact of the Study:  The changes in the PL of Ps. putida in the presence of TTAB and AlCl₃ indicate that different responses are utilized by bacteria to maintain optimal PL composition in the presence of such environmental pollutants.