Capillary electrophoresis of liposomes functionalized for protein binding

CE enabled assessing the attachment of hexa-histidine-tagged proteins to functionalized phospholipid liposomes. The liposomes were made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, phosphatidyl-ethanolamine, cholesterol and distearoyl-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol) in a molar ratio of 29:26:40:5. The unilamellar vesicles, which had an average diameter of 170 nm, were labelled by inclusion of FITC-dextran for fluorescence detection. CE was carried out in poly(vinyl alcohol) (PVA)-coated capillaries at 25 degrees C with a BGE consisting of Tris-HCl (50 mM, pH 8.0). For conjugation of the liposomes with the proteins (soluble synthetic receptor fragments with molecular mass of 60 and 70 kDa, respectively), Ni(2+) was implanted into the vesicle surface by an anchor lipid containing a nitrilotriacetate acid (NTA) group as complexation agent for the metal ions. The difference in surface charge enabled the separation of the different species of interest by CE: plain vesicles, vesicles functionalised with Ni-NTA, vesicle-protein complexes and the species formed upon removal of the Ni-ions by complexation with EDTA. Loss of the Ni-ions resulted in the release of the proteins and the reappearance of the plain Ni-free NTA-liposome species in the electropherograms.     

Interaction of ionic compounds with multilamellar liposomes. An electrokinetic model

We have proposed a theoretical model of interaction of ionic compounds (ionic adsorbate or ionic drugs), with multilamellar-liposomes, by means of their electrokinetic property variation with the ionic compound concentration. In this work, we show the complete development of the model proposed. Its theoretical results have been analysed to study the influence on the zeta-potential value of the following: number of membranes, size, shear-plane situation, critical concentration of formation (ccf), and the ionic compound concentration which annuls the zeta-potential value of the multilamellar-liposomes formed.     

Influence of structural factors on the enhanced activity of moxifloxacin: a fluorescence and EPR spectroscopic study

Partition coefficients of moxifloxacin in liposomes of dimyristoyl-L-α-phosphatidylcholine or dimyristoyl-L-α-phosphatidylglycerol and water were determined by spectrophotometry and fluorimetry. The K _p values obtained were larger than those reported for most of the other fluoroquinolones, a consequence of the structural changes observed in the molecule of moxifloxacin, which in turn change its acid/base properties. Introduction of a methoxy group at position 8 and a diazabicyclonyl ring at position 7 in the basic fluoroquinolone structure alters the charge distribution at the physiological pH of 7.4, and these changes seem to be responsible for its improved antibacterial potency and broader spectrum of activity. Location studies have also been performed using fluorescence and electron paramagnetic resonance (EPR) spectroscopies. The results show that moxifloxacin must be located near the phospholipid headgroups, similar to other fluoroquinolones, but contributions from a hydrophobic component were also detected. These results suggest that the enhanced activity of this drug may be related to a more facilitated entrance into the bacterial cell, perhaps including a mediator step involving electrostatic interaction with a hydrophobic component; this step then controls the extent or orientation of insertion and improves the electrostatic interaction.     

An in vitro feasibility study of controlled drug release from encapsulated nanometer liposomes using high intensity focused ultrasound

A liposome with a diameter ranging from 150 to 200 nm has been considered to be one of the optimal vehicles for targeted drug delivery in vivo since it is able to encapsulate drug and also circulate in the blood stream stably. Its small size, however, makes controlled release of its encapsulated content difficult. A feasibility study for applications of high intensity focused ultrasound (HIFU) of the mega-hertz frequency to induce controlled release of its content was carried out. This study, using the dynamic light scattering and transmission electron microscopic observation, demonstrated 21.2% of encapsulated fluorescent materials (FITC) could be released from liposomes with an average diameter of 210 nm when exposed to continuous (cw) ultrasound at 1.1 MHz (I_SPTA = 900 W/cm²) for 10 s and the percentage release efficiency can reach to 70% after 60 s irradiation. This result also reveals that rupture of relatively large liposomes (>100 nm) and generation of pore-like defects in the membrane of small liposomes (<100 nm) due to HIFU excitation might be the main causes of the release; the inertial cavitation took place during the irradiation. The controlled drug release from liposomes by HIFU may be proven to be a potential useful modality for clinical applications.     

Influence of cetyltrimethylammonium bromide on phosphatidylcholine-coated capillaries

Large unilamellar vesicles of egg-phosphatidylcholine (eggPC), a naturally occurring phospholipid, were used in capillary electrophoresis (CE) for semi-permanent coating of fused silica capillaries. The stability of the phospholipid coating was tested at different cetyltrimethylammonium bromide (CTAB) concentrations with and without CaCl₂ present in the coating solution. The effect of physical factors influencing the coating stability (e.g. duration of the coating time, storage temperature of the coating solution) were also studied. Standing overnight in background electrolyte (BGE) solution did not alter the eggPC phospholipid coating noticeably. The performance of the coating was tested with a mixture of basic proteins (lysozyme, ribonuclease A and -chymotrypsinogen A). Highest efficiencies (over 200,000 plates m^–1) were achieved when the capillary was filled for 15 h with a liposome solution containing both CTAB and CaCl₂.Electronic Supplementary Material Supplementary material is available for this article at     

Topical application of temoporfin-loaded invasomes for photodynamic therapy of subcutaneously implanted tumours in mice: a pilot study

Temoporfin (mTHPC) represents a very potent second-generation synthetic photosensitizer. It has shown to be effective in the photodynamic therapy of early or recurrent oral carcinomas, in the palliative treatment of refractory oral carcinomas and in the treatment of primary non-melanomatous tumours of the skin of the head and neck. Until now for all positive findings an intravenous application of the photosensitizer was mandatory. In the case of cutaneous malignant or non-malignant diseases a topical application of the drug onto the site of the disease followed by illumination, would be advantageous. Unfortunately, mTHPC is a highly hydrophobic drug with a low percutaneous absorption. The purpose of this experiment was to investigate the photodynamic efficacy of novel mTHPC-loaded invasomes after their topical application onto the skin of mice bearing the subcutaneously implanted human colorectal tumour HT29 followed by photoirradiation. Invasomes are vesicles containing in addition to phospholipids a mixture of terpenes (cineole, citral and d-limonene) or only one terpene (citral) and ethanol, as penetration enhancers. This was a pilot study since until now no data are available about the efficacy of mTHPC in the photodynamic therapy of HT29 tumours after its topical application. The aim of this experiment was to investigate whether a mTHPC-loaded invasome formulation can reduce tumour size by photodynamic therapy or at least to find a formulation slowing down tumour growth compared to the control group (mice without any treatment). The groups of mice treated with mTHPC–invasomes containing 1% of the terpene mixture prior to photoirradiation showed a significantly smaller (p < 0.05) tumour increase compared to control groups (mice without any treatment and mice only photoirradiated).     

Capillary electrophoresis method to determine siRNA complexation with cationic liposomes

Small interfering RNA (siRNA) inducing gene silencing has great potential to treat many human diseases. To ensure effective siRNA delivery, it must be complexed with an appropriate vector, generally nanoparticles. The nanoparticulate complex requires an optimal physiochemical characterization and the complexation efficiency has to be precisely determined. The methods usually used to measure complexation in gel electrophoresis and RiboGreen^® fluorescence‐based assay. However, those approaches are not automated and present some drawbacks such as the low throughput and the use of carcinogenic reagents. The aim of this study is to develop a new simple and fast method to accurately quantify the complexation efficiency. In this study, capillary electrophoresis (CE) was used to determine the siRNA complexation with cationic liposomes. The short‐end injection mode applied enabled siRNA detection in less than 5 min. Moreover, the CE technique offers many advantages compared with the other classical methods. It is automated, does not require sample preparation and expensive reagents. Moreover, no mutagenic risk is associated with the CE approach since no carcinogenic product is used. Finally, this methodology can also be extended for the characterization of other types of nanoparticles encapsulating siRNA, such as cationic polymeric nanoparticles.     

Chromatographic purification steps involved in the synthesis of prodrug-liposome-antibody-complexes

Summary The cytostatic effect of the widely used antitumor drug 1-β-D-arabinofuranosyl cytosine (ara C) can be improved by its chemical derivatization to lipophilic prodrugs. We have incorporated these prodrugs together with lipophilic biotin derivatives into membranes of unilamellar liposomes. Monoclonal antibodies were coupled to the biotin residues of the liposomes via avidin-biotin complexation resulting in prodrug-liposome-antibody complexes whichin vitro preferably bind to cells selectively recognized by the immobilized antibodies. The results open a promising way of drug targeting. The components and liposomal derivatives used for the stepwise preparation of the prodrug-liposome antibody complex are purified by means of preparative liquid chromatography. Lipophilic membrane components are chromatographed on silica gel, antibodies on hydroxylapatite and liposomal derivatives on Ultrogel AcA 22 columns. Concentration and desalting are achieved by ultrafiltration. The purification process can be quantitatively pursued by labelling with radioactive components.     

A comparative study of secondary ion yield from model biological membranes using Aun and C60 primary ion sources

Au_n⁺ and C₆₀⁺ primary ion sources have been used to acquire spectra from phospholipids, symmetric liposomes and asymmetric liposomes. We demonstrate that when using different ion beams different chemical information can be obtained. Symmetric and asymmetric liposomes, with 95% asymmetry, were produced and analysed with Au⁺, Au₃⁺ and C₆₀⁺ primary ion beams. C₆₀⁺ gave the greatest yield from the symmetric liposome but after correcting for the yield effects on the data obtained from the asymmetric liposome it has been shown that C₆₀⁺ is the most surface sensitive, providing the least information from the inner leaflet of the liposome. Au_n⁺ provides the greatest amount of information from the inner leaflet. The results present the possibility of designing ToF-SIMS experiments that selectively probe specific regions of a (bio)molecular surface.     

Cyclodextrins lessen the membrane damaging effect of nonionic tensides

Nonylphenyl-ethyleneoxide polymers containing 5, 9 and 30 ethyleneoxide groups per molecule build into the hydrophobic fatty acid chains of the cell membrane phospholipid dipalmitoyl-phosphatidylcholine (DPPC) resulting in a decreased main transition temperature, a decreased enthalpy of the main transition and in enhanced potassium permeability of DPPC liposomes. The -, - and -cyclodextrins form inclusion complexes with the tenzides lowering their free concentration. The complex formation lessens or sometimes totally prevents the membrane damaging effect of tensides. The effectivity order of cyclodextrins is CD>CD>CD.Presented at the Fourth International Symposium on Inclusion Phenomena and the Third International Symposium on Cyclodextrins, Lancaster, U.K. 20–25 July 1986.