Ganglioside–liposome immunoassay for the detection of botulinum toxin

A rapid and highly sensitive receptor immunoassay for botulinum toxin (BT) has been developed using ganglioside-incorporated liposomes. Botulism outbreaks are relatively rare, but their results can be very severe, usually leading to death from respiratory failure. To exert their toxicity, the biological toxins must first bind to receptors on the cell surface, and the trisialoganglioside GT1b has been identified as the cell receptor for BT. Therefore, in this study, GT1b was used to prepare the ganglioside–liposomes by spontaneous insertion into the phospholipid bilayer. In a sandwich-based, hybrid receptor immunoassay, BT is detected as a colored band on a nitrocellulose membrane strip, where BT bound to the GT1b-liposomes are captured by anti-BT antibodies immobilized in a band across the strip. The intensity of the colored band can be visually estimated, or measured by densitometry using computer software. The limit of detection (LOD) for BT in the lateral-flow assay system was 15 pg mL^–1, which is comparable to the limits of detection achieved with the most sensitive assays previously reported. However, this rapid assay can be completed in less than 20 min. These results demonstrate that the sandwich assay using GT1b-liposomes for detection of BT is rapid and very sensitive, suggesting the possibility for detecting BT in field screening, simply and reliably, without the need for complex instrumentation.     

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.     

A Biosensor Composed of Glucose Oxidase‐Containing Liposomes and MnO2‐Based Layered Nanocomposite

Glucose oxidase (GOD) was encapsulated in liposomes, and then the GOD‐containing liposomes were immobilized to a MnO₂‐based multilayered nanocomposite film grown electrochemically. Oxidation of glucose took place on the encapsulated GOD in the manganese oxide film, and the generated H₂O₂ molecules were oxidized catalytically at high valent Mn sites (4+) in the film. Anodic currents due to reoxidation of the reduced Mn ions (3+) were in proportion to the concentration of glucose from 19.6 to 107.1 mM. Such a simple construction of biosensor is applicable to a variety of combinations of liposomal enzymes and substrates.     

A Study of the Antioxidant Effect of Flavonic Compounds for Preventing Lipid Oxidation by Using Fluorescence Spectroscopy

In this paper a study of the effect of flavonic compounds in preventing and/or reducing the membrane lipid oxidation due to free radical attack was performed by using fluorescence spectroscopy techniques. Lipid peroxidation was investigated by using liposomes-artificial membrane models, which were prepared by lipid hydration method. Their oxidation was performed with a 15-W ultraviolet germicidal lamp having wavelength radiation 253.7 nm. In the first series of experiments, the protective effect of two synthetic antioxidants (quercetin and caffeic acid) against free radicals was monitored, while in the second series two 70% hydroalcoholic extracts from blueberry and blackcurrant leaves was used. It was determined that natural antioxidants have a much higher antioxidant power against free radicals than synthetic compounds but they degrade after two hours of oxidation. Liposomes are better protected when using natural antioxidants, but their degradation is completed more quickly, than in the case of synthetic antioxidants.     

Electric impedance method for evaluation of the release property of calcein-encapsulated liposomes

This paper is concerned with the study on development of a novel method for evaluation of the liposomes release property by measuring the electric impedance changes of liposome suspensions. Calcein/NaOH encapsulated liposomes (calcein-liposomes) were prepared with deionized water and were treated with ultrasonic irradiation in order to investigate the release property of the liposomes. To validate the proposed impedance measuring method, the calcein release rates were evaluated both by the impedance changes and the fluorescence intensity changes in calcein-liposome suspensions. With the comparison of these results obtained by the two methods, it is shown that the impedance method has much wider detecting concentration range than the fluorescence one. Furthermore, the impedance method can be efficiently used for evaluation of the release property on various ionic substances encapsulated within liposomes.     

Preparation approaches of the coated capillaries with liposomes in capillary electrophoresis

The use of liposomes as coating materials in capillary electrophoresis has recently emerged as an important and popular research area. There are three preparation methods that are commonly used for coating capillaries with liposomes, namely physical adsorption, avidin–biotin binding and covalent coupling. Herein, the three different coating methods were compared, and the liposome-coated capillaries prepared by these methods were evaluated by studying systematically their EOF characterization and performance (repeatability, reproducibility and lifetime). The amount of immobilized phospholipids and the interactions between liposome or phospholipid membrane and neutral compounds for the liposome-coated capillaries prepared by these methods were also investigated in detail. Finally, the merits and disadvantages for each coating method were reviewed.     

Investigation of NeutrAvidin-tagged liposomal nanovesicles as universal detection reagents for bioanalytical assays

Ligand-tagged liposomes, obtained by covalent conjugation of ligands to the liposomal surface, have been widely used as detection reagents in bioanalytical assays. A non-covalent conjugation method where IgG was attached to protein G-tagged liposomes has been recently utilized. To enlarge the application of non-covalent methods to a greater variety of ligands, including peptides, proteins, and nucleic acids, we developed and optimized a new method for the preparation of NeutrAvidin-tagged liposomes with subsequent attachment of biotinylated ligands. Two assays were used to investigate the feasibility of NeutrAvidin-tagged liposomes. The first assay was a competitive immunoassay for detecting rabbit antibodies, while the second assay was a sandwich hybridization assay for detecting a synthetic target: a DNA fragment of Erwinia amylovora. To produce the immunoliposomes for the detection of rabbit IgG, NeutrAvidin was covalently tagged to the liposomal surface at four different starting molar percentages (0.1, 0.2, 0.4, and 0.8). The biotinylated goat anti-rabbit IgG at three different molar ratios of biotin to IgG (5, 10, and 20) were then attached to the NeutrAvidin-tagged liposomes by using two different molar ratios of goat anti-rabbit IgG to NeutrAvidin (1 and 5). After the comparison of all 24 combinations, the best result was obtained with the 0.1 starting molar percentage of NeutrAvidin, 20 as the molar ratio of biotin to goat IgG, and 1 as molar ratio of IgG to NeutrAvidin. Under these optimized conditions, the limit of detection (LOD) for rabbit IgG was 38 pmol/mL. Moreover, the best combination for the sandwich hybridization assay was with the 0.1 starting molar percentage of NeutrAvidin-tagged liposomes and when the molar ratio of biotinylated reporter probe to NeutrAvidin was equal to 1. The LOD for the synthetic target DNA fragment of E. amylovora was ca. 30 pmol/mL. Both assays could be completed in about 30 min without the requirement of sophisticated equipment or techniques. Therefore, these two assays have successfully demonstrated the feasibility of NeutrAvidin-tagged liposomal nanovesicles as a universal reagent for the attachment of different types of biotinylated ligands in a fast and easy coupling process. In addition, these ligand-tagged liposomes have the potential for wide use in different types of bioanalytical assays.     

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.     

Lipophilic nucleic acids — A flexible construction kit for organization and functionalization of surfaces

Lipophilic nucleic acids have become a versatile tool for structuring and functionalization of lipid bilayers and biological membranes as well as cargo vehicles to transport and deliver bioactive compounds, like interference RNA, into cells by taking advantage of reversible hybridization with complementary strands. This contribution reviews the different types of conjugates of lipophilic nucleic acids, and their physicochemical and self-assembly properties. Strategies for choosing a nucleic acid, lipophilic modification, and linker are discussed. Interaction with lipid membranes and its stability, dynamic structure and assembly of lipophilic nucleic acids upon embedding into biological membranes are specific points of the review. A large diversity of conjugates including lipophilic peptide nucleic acid and siRNA provides tailored solutions for specific applications in bio- and nanotechnology as well as in cell biology and medicine, as illustrated through some selected examples.