Native Nano-electrospray Differential Mobility Analyzer (nES GEMMA) Enables Size Selection of Liposomal Nanocarriers Combined with Subsequent Direct Spectroscopic Analysis
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Gas-phase electrophoresis employing a nano-electrospray differential mobility analyzer (nES DMA), aka gas-phase electrophoretic mobility molecular analyzer (nES GEMMA), enables nanoparticle separation in the gas-phase according to their surface-dry diameter with number-based concentration detection. Moreover, particles in the nanometer size range can be collected after size selection on supporting materials. It has been shown by subsequent analyses employing orthogonal methods, for instance, microscopic or antibody-based techniques, that the surface integrity of collected analytes remains intact. Additionally, native nES GEMMA demonstrated its applicability for liposome characterization. Liposomes are nanometer-sized, biodegradable, and rather labile carriers (nanoobjects) consisting of a lipid bilayer encapsulating an aqueous lumen. In nutritional and pharmaceutical applications, these vesicles allow shielded, targeted transport and sustained release of bioactive cargo material. To date, cargo quantification is based on bulk measurements after bilayer rupture. In this context, we now compare capillary electrophoresis and spectroscopic characterization of vesicles in solution (bulk measurements) to the possibility of spectroscopic investigation of individual, size-separated/collected liposomes after nES GEMMA. Surface-dried, size-selected vesicles were collected intact on calcium fluoride (CaF2) substrates and zinc selenide (ZnSe) prisms, respectively, for subsequent spectroscopic investigation. Our proof-of-principle study demonstrates that the off-line hyphenation of gas-phase electrophoresis and confocal Raman spectroscopy allows detection of isolated, nanometer-sized soft material/objects. Additionally, atomic force microscopy-infrared spectroscopy (AFM-IR) as an advanced spectroscopic system was employed to access molecule-specific information with nanoscale lateral resolution. The off-line hyphenation of nES GEMMA and AFM-IR is introduced to enable chemical imaging of single, i.e., individual, liposome particles.Keywords:
Ion-mobility spectrometry
Various kinds of long-circulating liposome, such as ganglioside GM1-, polyethyleneglycol- (PEG-), and glucuronide-modified liposomes, have been developed for passive targeting of liposomal drugs to tumours. To evaluate the in vivo behaviour of such long-circulating liposomes, we investigated the liposomal trafficking, especially early trafficking just after injection of liposomes, by a non-invasive method using positron emission tomography (PET). Liposomes composed of dipalmitoylphosphatidylcholine, cholesterol, and modifier, namely, GM1, distearoylphosphatidylethanolamine (DSPE)–PEG or palmityl-D-glucuronide (PGlcUA), were labelled with [2-18F]-2-fluoro-2-deoxy-D-glucose ([2-18F]FDG), and administered to mice bearing Meth A sarcoma after having been sized to 100 nm. A PET scan was started immediately after injection of liposomes and continued for 120 min. PET images and time–activity curves indicated that PEG liposomes and PGlcUA liposomes were efficiently accumulated in tumour tissues time dependently from immediately after injection. In contrast, GM1 liposomes accumulated less in the tumour as was also the case for control liposomes that contained dipalmitoylphosphatidylglycerol (DPPG) instead of a modifier. Long-circulating liposomes including GM1 liposomes, however, remained in the blood circulation and avoided liver trapping compared with control DPPG liposomes. These data suggest that PGlcUA and PEG liposomes start to accumulate in the tumour just after injection, whereas GM1 liposomes may accumulate in the tumour after a longer period of circulation.
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The liposome particle size is an important parameter because it strongly affects content release from liposomes as a result of different bilayer curvatures and lipid packing. Earlier, we developed pH-responsive polysaccharide-derivative-modified liposomes that induced content release from the liposomes under weakly acidic conditions. However, the liposome used in previous studies size was adjusted to 100-200 nm. The liposome size effects on their pH-responsive properties were unclear. For this study, we controlled the polysaccharide-derivative-modified liposome size by extrusion through polycarbonate membranes having different pore sizes. The obtained liposomes exhibited different average diameters, in which the diameters mostly corresponded to the pore sizes of polycarbonate membranes used for extrusion. The amounts of polysaccharide derivatives per lipid were identical irrespective of the liposome size. Introduction of cholesterol within the liposomal lipid components suppressed the size increase in these liposomes for at least three weeks. These liposomes were stable at neutral pH, whereas the content release from liposomes was induced at weakly acidic pH. Smaller liposomes exhibited highly acidic pH-responsive content release compared with those from large liposomes. However, liposomes with 50 mol% cholesterol were not able to induce content release even under acidic conditions. These results suggest that control of the liposome size and cholesterol content is important for preparing stable liposomes at physiological conditions and for preparing highly pH-responsive liposomes for drug delivery applications.
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The uptake mechanisms of liposomes by rat peritoneal macrophages (PMs) were investigated. Incubation of liposomes with fresh rat serum enhanced the uptake of liposomes depending on the liposome size and cholesterol (CH) content. The binding of liposomes was also enhanced by serum, and this increase depended on the size and CH content as in the case of liposome uptake, which suggested that the binding of opsonized liposomes with PMs govern the extent in liposome uptake. The rate constant for the internalization (kint) was calculated by measuring both uptake and binding. The kint cannot explain the variation of liposome uptake for different sizes and CH contents. The kint values for liposomes with high (44%) and medium (33%) CH contents were constant (2.5-1), while those for liposomes with low (22%) Ch content were significantly elevated (5-9h-1). These results indicate the persence of at least two kinds of uptake mechanisms of liposomes. Treatment of serum with anti-C3 antiobdy completly inhibited the enhanced uptake of CH-high, large liposomes, which suggested that complement receptor-mediated phagocytosis may be an uptake mechanism for CH-high and -medium liposomes. In addition, complement-independent enhanced uptake was suggested for CH-low liposomes, since no inhibition was observed for CH-low liposomes by anti-C3 antibody and these liposomes were disintegrated in serum via complement-independent pathway. These results provided evidence that PMs take up liposomes via complement-dependent and indepenedent mechanisms depending on the CH content of the liposomes.
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This paper reports a new method for the direct determination of SO2in HMs using fast field asymmetric-wave ion mobility spectrometry (FAIMS) coupled with a headspace air bubbling method.
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• FAIMS-MS has been used for the analysis of non-covalent complexes formed by 3-MX. The singly charged (3-MX) n (n = 4-12) complexes show maximum FAIMS transmission at different CF values, with the optimum CF decreasing as the size of the cluster increases (Fig. 4). • 3-Methylxanthine was prepared as a 0.5 mM solution in 60:40 methanol:water with 1 mM ammonium acetate, or with 1 mM sodium hydroxide, to promote the formation of 3-MX clusters with Na + , and to enable the detection of higher-ordered clustered 3-MX structures. • 3-MX solutions were analysed by FAIMS using an Agilent 6230 TOF MS (Agilent Technologies) with a Jet Stream ESI source, combined with a prototype miniaturised chip-based FAIMS device (Owlstone Ltd., Cambridge), located in front of the mass spectrometer inlet capillary (Fig. 2). The FAIMS device consists of multiple planar electrode channels each with a 100 µm gap and an electrode length of 700 µm. • The TOF MS experimental conditions in positive ion mode were: drying gas: 8 L/min at 150 °C; sheath gas: 10 L/min at 200 °C; nebuliser gas: 30 psig; capillary voltage: 3.5 kV; nozzle voltage: 2 kV; fragmentor voltage: 150-250 V; and a sample flow rate of 10 µL/min using a syringe pump. The optimum FAIMS conditions for the selective transmission of the different 3- MX clusters, singly, doubly and multiply charged species, were determined by conducting a compensation field (CF) sweep from -2 to 5 Td at a rate of 0.5 Td/sec, for dispersion fields (DF) in the range 194 to 323 Td. Conclusions • Hyphenation of FAIMS-MS and IM-MS has been used for the analysis of 3-MX complexes. • This preliminary study into the structural analysis of 3-MX complexes shows a complexity of non-covalently clustered structures. • FAIMS selection has been used for the separation of overlapping charge states of 3-MX complexes. • Increased S:N ratio is observed for higher-order 3-MX complexes using FAIMS-MS. • 3-MX singly charged complexes formed in the presence of sodium show different CF values
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Part 1 General Introduction to Liposomes: 1. Chemistry of lipids and liposomes. 2. Structure of amphiphilic aagregates. 3. Preparation of liposomes. 4. Mechanism of liposome formation. 5. Liposome characterization methods. Part 2 Applications of Liposomes in Basic Sciences: 6. Applications of liposomes in theoretical sciences. 7. Applications in biophysics. 8. Liposomes in the studies of the evolution of life. 9. Applications in chemistry. 10. Reconstitution of proteins. Part 3 Applications of Liposomes in Pharmacology and Medicine: 11. Liposomes as a drug delivery system. 12. Liposomes in the treatment of infectious diseases. 13. Liposomes as immunoadjuvants. 14. Liposomes in anticancer therapy. 15. Liposomes and inflammations. 16. Other medical applications of liposomes. 17. Other administration routes of liposomes. 18. Site specific drug delivery. Part 4 Other Applications of Liposomes: 19. Cosmetic applications of liposomes. 20. Liposomes as a carrier system in genetic engineering. 21. Liposomes in diagnostics. 22. Liposomes in food industry. 23. Liposomes in ecology and other applications. 24. Industrial manufacturing of liposomes.
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In order to reveal quantitatively interaction of complement (C) system with liposomes, we determined C3 fragments associated with the liposomes having different sizes after incubation in human plasma. The amount of C3 fragments per unit surface area on the unstable liposomes (Man-liposomes) which modified with synthesized glycolipid (cetylmannoside, Man) increased with the increase in the liposome size, whereas that of C3 fragments on the stable ones (PC-liposomes) was little found. In addition, the instability of Man-liposomes also increased with the increase in the liposome size and there was linear correlation. On the other hand, the amount of bound plasma proteins per unit surface area of liposomes was approximately constant regardless of differences of lipid composition and size, and showed no correlation with instability of the liposomes in the plasma. These in vitro results indicate that the instability of Man-liposomes is governed by the affinity of C system and the C system can recognize not only liposome surface characteristics but also liposome sizes. To demonstrate clearly the reason for the linear correlation between the affinity of C system and liposome size, we discussed the underlying mechanism based on the previous finding that the activation of C system by Man-MLVs was enhanced through classical C pathway and presented the hypothesized osculating model on the assumption that extent of C activation is governed by attachment of a C activator.
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