Asymmetric flow field‐flow fractionation of liposomes: 2. Concentration detection and adsorptive loss phenomena

The applicability of different concentration detection methods for online quantification of liposomes upon asymmetric flow field‐flow fractionation was investigated. Filter‐extruded egg phosphatidylcholine liposomes of different size were used. Online quantification using a differential refractive index (dRI) detector was found feasible for relatively high sample loads in the magnitude of 100 μg lipid (under the chosen fractionation conditions). UV–Vis detection of the turbidity of liposomes was ruled out as online detection method because turbidity increases with particle size and the signal is not only concentration but also particle‐size dependent. Staining of liposomes by Rhodamine phosphatidylethanolamine or Sudan Red and subsequent online UV–Vis detection at the absorption maximum of the dye enabled quantification with much higher sensitivity than dRI detection. Furthermore analyte loss and carry‐over phenomena upon repeated injection of varying liposome sample loads were studied using regenerated cellulose (RC) membranes as accumulation wall. It could be shown that RC membranes are prone to adsorption in case of very small sample loads (0.5 μg). This effect may be overcome by pre‐saturation of the membrane with sample loads of at least 2 μg. For higher sample loads adsorptive losses play a minor role. Recovery from pre‐saturated membranes reached approximately 100% and carry‐over was found negligible.     

Studying phase separation in confinement

Cells organize their interior through membrane-bound organelles and through membraneless condensates that are formed by liquid–liquid phase separation (LLPS). The complex process of coacervation that is involved in LLPS is challenging to study in living cells. Hence, studying coacervation in cell-mimicking synthetic containers can yield valuable insights. Here, we review recent progress with respect to studying LLPS (particularly coacervation) in artificial compartments, from water-in-oil droplets to membranous liposomes. We describe different strategies to form and control coacervates in microconfinements and to study their physicochemical and biological characteristics. We also describe how coacervation can itself be used in container formation. This review highlights the importance of in vitro coacervate studies for understanding cellular biology and for designing synthetic cells.     

Protein-induced leakage and membrane destabilization of phosphatidylcholine and phosphatidylserine liposomes

By using spectroscopic and colloidal chemistry methods we studied the interactions of globular proteins with phospholipid membranes in relation to protein-promoted membrane fusion. We considered the effect of protein sorption on the destabilization of phosphatidylcholine and phosphatidylserine liposome membranes. Experimentally, we injected the proteins into fluorophore-quencher embedded liposome dispersions and recorded the leakage of fluorophore-quencher from the liposomes' inner compartment, which is due to the protein-induced destabilization of the phospholipid membranes. The release of fluorophore-quencher strongly depends on the protein concentration. The existence of monovalent and polyvalent cations also influences the protein-induced membrane destabilization by affecting the hydrophobic and electrostatic interactions.     

Effects of liposomal phophatidylserine on phagocytic uptake of liposomes by macrophage-like HL-60RG cells

The purpose of this work is to know the effect of surface properties of liposomes on their phagocytic uptake by macrophages. For this, liposomes were prepared by the Bangham technique from the mixture of phosphatidylcholine (PC) and cholesterol (Chol) incorporated either with phosphatidylserine (PS), phosphatidylethanolamine (PE) or phosphatidic acid (PA). The liposomes thus prepared had diameters in the range between 150 and 260 nm. Electric surface properties of the liposomes and the macrophages differentiated from HL-60RG cells were determined by measuring their electrophoretic mobilities. The phagocytic uptake of liposomes with different contents of PS, PE and PA by macrophage-like HL-60RG cells was investigated by measuring oxygen consumption associated with phagocytic uptake. The phagocytic activity was found to be the highest with the PC–Chol liposomes containing 7 mol% PS, but no significant effects were observed with PA- and PE-containing PC–Chol liposomes. As the uptake was independent of the electric surface property of liposomes, PS was concluded to be specifically important for phagocytic activity of macrophages.     

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.     

Cholera toxin subunit B detection in microfluidic devices

Fluorescence and electrochemical microfluidic biosensors were developed for the detection of cholera toxin subunit B (CTB) as a model analyte. The microfluidic devices were made from polydimethylsiloxane (PDMS) using soft lithography from silicon templates. The polymer channels were sealed with a glass plate and packaged in a polymethylmethacrylate housing that provided leakproof sealing and a connection to a syringe pump. In the electrochemical format, an interdigitated ultramicroelectrode array (IDUA) was patterned onto the glass slide using photolithography, gold evaporation and lift-off processes. For CTB recognition, CTB-specific antibodies were immobilized onto superparamagnetic beads and ganglioside GM₁ was incorporated into liposomes. The fluorescence dye sulforhodamine B (SRB) and the electroactive compounds potassium hexacyanoferrate (II)/hexacyanoferrate (III) were used as detection markers that were encapsulated inside the liposomes for the fluorescence and electrochemical detection formats, respectively. Initial optimization experiments were carried out by applying the superparamagnetic beads in microtiter plate assays and SRB liposomes before they were transferred to the microfluidic systems. The limits of detection (LoD) of both assay formats for CTB were found to be 6.6 and 1.0 ng mL⁻¹ for the fluorescence and electrochemical formats, respectively. Changing the detection system was very easy, requiring only the synthesis of different marker-encapsulating liposomes, as well as the exchange of the detection unit. It was found that, in addition to a lower LoD, the electrochemical format assay showed advantages over the fluorescence format in terms of flexibility and reliability of signal recording.     

Fluorescence study of the dynamic interaction between E1(145–162) sequence of hepatitis GB virus C and liposomes

The physicochemical characterization of the peptide sequence E1(145–162) corresponding to the structural protein E1 of the hepatitis G virus was done by studying its interaction with model membranes. Small unilamellar vesicles (SUVs) of dimyristoylphosphatidylglycerol or dimyristoylphosphatidylcholine were chosen as mimetic membranes. Peptide incorporation and location in the phospholipid bilayer was investigated by fluorescence anisotropy with SUVs labeled with diphenylhexatriene (DPH) or trimethylammonium–DPH. The addition of the peptide E1(145–162) showed significant changes in the anisotropy values of the probe located at the air/water interface. These results indicate that the peptide E1(145–162) preferably interacts with the lipid surface without penetrating inside the bilayer. A series of fluorescence experiments based on tryptophan peptide fluorescence were modeled by means of multivariate curve resolution-alternating least squares (MCR-ALS) algorithm to further study the peptide interaction with bilayers at different temperatures. The preliminary results obtained with MCR-ALS showed how the peptide concentration decay is directly linked to the appearance of a new specie, which corresponds to the lipid-peptide binding. These results provide useful information for the design of synthetic immunopeptides that can be incorporated into a liposomal system with potential to promote a direct delivery of the membrane-incorporated immunogen to the immunocompetent cells, thus increasing the immuno response from the host. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.