Use of a novel microfluidic disk in the analysis of single-cell viability and the application to Jurkat cells

Jurkat cells were trapped in the microchambers of a novel disk-shaped cell separation device and stained with Cellstain. Approximately 90% of the cells were living. Single cells were isolated with a branching microchannel after rotation at 4500rpm for 30s, demonstrating that a living single cell could be trapped in the microchambers.     

Size and Surface Charge Effect of 5-Aminolevulinic Acid-Containing Liposomes on Photodynamic Therapy for Cultivated Cancer Cells

5-Aminolevulinic acid (ALA)-containing liposomes having various average diameters and/or positive surface charges were prepared, and their photodynamic therapy (PDT) efficacy for murine thymic lymphoma cells, EL-4 cells, cultivated in vitro was investigated. The PDT efficacy for EL-4 cells and the accumulation of ALA-induced protoporphyrin IX (PpIX) in the cells increased with a decrease in the average diameter of liposomes. In particular, the ALA-containing liposomes smaller than 63.5 nm in diameter promoted the PDT efficacy in comparison with that of ALA alone. We also found no significant changes in PDT efficacy and PpIX accumulation with increasing positive surface charges of liposomes.     

Absorption and emission spectral shifts of rose bengal associated with DMPC liposomes

Rose bengal is a water-soluble xanthene dye that is currently used in ophthalmology for the diagnosis of dry eyes. Although the dye is also a potential photosensitizer for photodynamic therapy of tumors, owing to insufficient lipophilicity and tumor accumulation, the clinical application of rose bengal in photodynamic therapy has been hampered. Liposomal encapsulation was seen as a promising approach to overcome these disadvantages, to which end, the spectral properties of the dye in the presence of materials for liposome preparation were studied. The presence of phospholipid influenced the spectral properties of the dye, probably due to the establishment of an equilibrium between monomeric and dimeric forms of the dye, since the photophysical properties of rose bengal depend strongly on its environment. The liposomal encapsulation of the dye generates stronger emission than the free form of the colorant; increased lipid:dye ratio further enhances this emission.     

Physicochemical characteristics of liposomes formed with internal wool lipids

The bilayer-forming capability of internal wool lipids and their physicochemical properties were studied in an attempt to enhance our understanding of the lipid structure, present in wool and other keratinized tissues. Internal wool lipids were extracted and analyzed, and the mixture obtained [sterol esters (10%), free fatty acids (24%), sterols (11%), ceramides (46%), and cholesteryl sulfate (9%)] was shown to form stable liposomes. A phase-transition temperature of 60°C was obtained from nuclear magnetic resonance spectra for this lipid mixture. The spontaneous permeability of these vesicles was lower than that of phosphatidylcholine liposomes but slightly higher than that of the vesicles formed with lipids extracted from other keratinized tissues with higher amounts of cholesterol. The transmission electron micrographs showed large vesicular aggregates of approximately 300 nm, which seem to be made up of smaller structures of approximately 20 nm in size. This particular structure could account for the large diameters and small internal volumes found by dynamic light-scattering and spectrofluorometric measurements.     

Preparation of liposomes using the supercritical anti-solvent (SAS) process and comparison with a conventional method

Two methods to produce liposomes encapsulating a fluorescent marker were compared: the supercritical anti-solvent (SAS) method and a conventional one (Bangham). Liposome size and encapsulation efficiency were measured to assess the methods. Micronized lecithin produced by the SAS process was characterized in terms of particle size, morphology and residual solvent content in order to investigate the influence of experimental parameters (pressure, CO₂/solvent molar ratio and solute concentration). It appears that when the lecithin concentration increases from 15 to 25 wt.%, at 9 MPa and 308 K, larger (20-60 μm) and less aggregated lecithin particles are formed. As concerns liposomes formed from SAS processed lecithin, size distribution curves are mainly bimodal, spreading in the range of 0.1-100 μm. Liposome encapsulation efficiencies are including between 10 and 20%. As concerns the Bangham method, more dispersed liposomes were formed; encapsulation efficiencies were about 20%, and problems of reproducibility have been raised.     

Liposome encapsulation protects bacteriocin-like substance P34 against inhibition by Maillard reaction products

Bacteriocins are biopreservatives with potential to be used in heat processed foods, and liposome encapsulation may offer a protective effect against interaction of these peptides with food compounds. In this work, Maillard reaction products (MRPs) were prepared by heating (90 °C for 7 h) different combinations of sugars and amino acids, and their inhibitory capacity against free and liposome-encapsulated bacteriocin-like substance (BLS) P34 was evaluated. MRPs formed by fructose and glycine had the highest inhibitory capacity against free and encapsulated BLS P43, remaining 50% of initial activity. Liposome encapsulation increased stability of BLS P34 when it was confronted to glucose/glycine, galactose/glycine, sucrose/glycine and glucose/alanine MRPs. Inhibition mostly occurs within the first 24 h of incubation of BLS P34 and MRPs. Inhibitory activity seems to be associated to melanoidin formation and antioxidant capacity of MRPs. The results show that liposome encapsulation of BLS P34 may be an interesting technique to increase the stability of antimicrobial peptides in heat treated foods.     

Catalytic mechanisms of metmyoglobin on the oxidation of lipids in phospholipid liposome model system

The catalytic mechanism of metmyoglobin (metMb) on the development of lipid oxidation in a phospholipid liposome model system was studied. Liposome model system was prepared with metMb solutions (2.0, 1.0, 0.5, and 0.25 mg metMb/mL) containing none, diethylenetriamine pentaacetic acid (DTPA), desferrioxamine (DFO), or ferric chloride and lipid oxidation was determined at 0, 15, 30, 60, and 90 min of incubation at 37 °C. Metmyoglobin catalysed lipid oxidation in the liposome system, but the rate of lipid oxidation decreased as the concentration of metMb increased. The amount of free ionic iron in the liposome solution increased as the concentration of metMb increased, but the rate of metMb degradation was increased as the concentration of metMb decreased. The released free ionic iron was not involved in the lipid oxidation of model system because ferric iron has no catalytic effect without reducing agents. Both DFO and DTPA showed antioxidant effects, but DFO was more efficient than DTPA because of its multifunctional antioxidant ability as an iron and haematin chelator and an electron donor. The antioxidant activity of DTPA in liposome solution containing 0.25 mg metMb/mL was two times greater than that with 2 mg metMb/mL due to the increased prooxidant activity of DTPA-chelatable compounds. It was concluded that ferrylmyoglobin and DTPA-chelatable haematin generated from the interaction of metMb and LOOH, rather than free ionic iron, were the major catalysts in metMb-induced lipid oxidation in phospholipid liposome model system.     

Antilisterial activity and stability of nanovesicle-encapsulated antimicrobial peptide P34 in milk

Bacillus sp. P34, a strain isolated from aquatic environments of Brazilian Amazon basin, produces a bacteriocin-like substance (BLS) which was encapsulated in nanovesicles prepared from partially purified soy lecithin. The efficiency of free and encapsulated BLS P34 to control the development of L. monocytogenes and maintenance of antimicrobial activity was assessed over time in milk. The antimicrobial activity of free and encapsulated BLS P34 decreased approximately 50% after 4 days of storage (<4 °C) in skim and whole milk. After this period there was not significant loss of activity up to 21 days. The viable counts of Listeria monocytogenes in skim and whole milk containing 3200 AU/ml of free or encapsulated BLS P34 were always lower than those observed in controls without bacteriocin at both 30 °C and 7 °C. At 1600 AU/ml concentration, free and encapsulated BLS P34 were inhibitory to L. monocytogenes in skim milk, when compared with the control at 7 days. Nanovesicle-encapsulated and free BLS P34 shows potential use as biopreservative for application in milk-derived products.     

The specific antibacterial activity of liposome-encapsulated Clove oil and its application in tofu

In this study, the antibacterial activities of Clove oil and liposome-encapsulated Clove oil were investigated. First, the antibacterial activity of Clove oil demonstrated that the essential oil exhibited favorable antimicrobial activity for both Escherichia coli and Staphylococcus aureus. However, a setback of using Clove oil as a disinfectant is its low chemical stability. Then Clove oil was incorporated into a liposome formulation to increase its stability. The optimal polydispersity index (PDI) (0.196), Zeta potential (−24.5 mV) and entrapment efficiency (20.41%) of liposome were obtained at the concentration of Clove oil to 5.0 mg/mL. In addition, selective antimicrobial activity for S. aureus by utilizing pore-forming toxins (PFTs) to activate Clove oil release from liposome was observed. By contrast, liposome-encapsulated Clove oil has no effect on E. coli that doesn't secrete PFTs because antimicrobial component can't reach bacteria. Gas chromatography (GC) assay found that when liposome met S. aureus that secrete PFTs, PFTs would insert into the liposome membranes and form pores, through which the encapsulated Clove oil was released. Besides, liposome-encapsulated Clove oil exhibited efficient antimicrobial activity for S. aureus in tofu.     

精子干细胞转染法制备转基因兔的研究

采用直接注射的方法 ,将脂质体包裹的含人乳铁蛋白基因重组质粒 pLNCXHLF注入兔睾丸组织中 ,一个月后与正常雌兔交配。所产仔兔经PCR、Southern检测证明获得了转基因兔 ,转基因阳性率平均为 35 %。实验结果表明 ,通过注射可将外源基因导入到曲细精管中 ,进而完成对精子干细胞的转染 ,获得携带外源基因的成熟精子 ,受精后可得到转基因动物。该方法是一种简捷、有效的新途径 ,既节省时间又降低了成本 ,为利用精子载体制备转基因动物的研究提供了很有价值的参考。