Vesicle size and stability of biomimetic liposomes from 3'-sulfo-Lewis a (SuLea) containing glycolipids

We report on the use of a natural Lewis type saccharide ligand, 3′-sulfo-Lewis a (SuLe^a) for glycocalyx-mimetic surface modification of liposomes. Two SuLe^a-containing glycolipids, monovalent SuLe^a-lipid and trivalent SuLe^a (TSuLe^a)-lipid, were synthesized, and used with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol to prepare unilaminar vesicles (ULVs) by a freeze–thaw and extrusion method. The effects of the glycolipid concentrations and the pore sizes of extrusion membranes on vesicle size and stability were investigated by photon correlation spectroscopy (PCS). Glycoliposomes, with 5% SuLe^a- or TSuLe^a-lipids obtained by 50 nm extrusion, had 25–30% more vesicles less than 100 nm in diameter compared with the 100 nm extrusion. TSuLe^a-liposomes always produced larger vesicle size than SuLe^a-liposomes, which we attribute to the larger TSuLe^a headgroup. Both SuLe^a- and TSuLe^a-liposomes increased their vesicle size with increasing glycolipid concentration from 5% to 15%, and demonstrated good stability at room temperature for over 1 month. Further increasing the glycolipid concentration to 20% resulted in large vesicle aggregation after 5 days for TSuLe^a-liposomes, while the SuLe^a-liposomes remained stable for 10 days. SuLe^a- and TSuLe^a-liposomes with 15% glycolipids demonstrated better stability due to the electrostatic effect from the negatively charged SuLe^a and TSuLe^a headgroups. The results indicate that the biomimetic liposomes with SuLe^a- and TSuLe^a-lipids with 5 to 15% incorporation are sufficiently stable for the potential applications in targeted drug delivery.     

Effect of molecular weight in amphipathic polyethyleneglycol on prolonging the circulation time of large unilamellar liposomes

The effect in mice of the molecular weight of polyethyleneglycol on prolonging the circulation time of large unilamellar liposomes (LUVs) was examined using four different distearoyl N-(monomethoxy polyethyleneglycol succinyl) phosphatidylethanolamines (DSPE-PEGs). The molecular weights tested were 1000, 2000, 5000 and 12000. Incorporation of 6 mol% of DSPE-PEG in LUV composed of distearoylphosphatidylcholine (DSPC) / cholesterol (CH) (1:1 in molar ratio) increased the blood circulation half-life significantly more than those without DSPE-PEG derivatives. DSPE-PEGs with molecular weights of 1000 and 2000 prolonged the circulation time of liposomes more than other DSPE-PEGs with higher molecular weights, such as 5000 and 12000. Their effects are also higher than ganglioside GM1, a well described glycolipid with this effect. DSPC/CH LUV-incorporated DSPE-PEG with a molecular weight of 2000 displayed a high concentration in the blood, approximately 40% of the dose, 6 h after the injection.     

Sequence-specific binding of DNA to liposomes containing di-alkyl peptide nucleic acid (PNA) amphiphiles

We present a method to covalently attach peptide nucleic acid (PNA) to liposomes by conjugation of PNA peptide to charged amino acids and synthetic di-alkyl lipids ("PNA amphiphile," PNAA) followed by co-extrusion with disteroylphosphatidylcholine (DSPC) and cholesterol. Attachment of four Glu residues and two ethylene oxide spacers to the PNAA was required to confer proper hydration for extrusion and presentation for DNA hybridization. The extent of DNA oligomer binding to 10-mer PNAA liposomes was assessed using capillary zone electrophoresis. Nearly all PNAs on the liposome surface are complexed with a stoichiometric amount of complementary DNA 10-mers after 3-h incubation in pH 8.0 Tris buffer. No binding to PNAA liposomes was observed using DNA 10-mers with a single mismatch. Longer DNA showed a greatly attenuated binding efficiency, likely because of electrostatic repulsion between the PNAA liposome double layer and the DNA backbone. Langmuir isotherms of PNAA:DSPC:chol monolayers indicate miscibility of these components at the compositions used for liposome preparation. PNAA liposomes preserve the high sequence-selectivity of PNAs and emerge as a useful sequence tag for highly sensitive bioanalytical devices.     

Interactions of cyclodextrins with dipalmitoyl, distearoyl, and dimyristoyl phosphatidyl choline liposomes. A study by leakage of carboxyfluorescein in inner aqueous phase of unilamellar liposomes

The interaction of cyclodextrins (CDs) with L-alpha-dipalmitoyl phopsatidyl choline (DPPC), L-alpha-distearoyl phosphatidyl choline (DSPC), and L-alpha-dimyristoyl phosphatidyl choline (DMPC) unilamellar liposomes was investigated by the leakage of carboxylfluorescein (CF) entrapped in the inner aqueous phase of liposomes, at 25 degrees C (DPPC and DSPC liposomes) and at 5 degrees C (DMPC liposomes). The efficiency of CDs for CF leakage was remarkable in the order of heptakis (2,6-di-O-methyl)-beta-CD (DOM-beta-CD) > alpha-CD > heptakis (2,3,6-tri-O-methy)-beta-CD (TOM-beta-CD) from DPPC liposomes, in the order of DOM-beta-CD > TOM-beta-CD > alpha-CD from DSPC liposomes and in the order of alpha-CD > DOM-beta-CD > TOM-beta-CD from DMPC liposomes. The other CDs used in the present studies, beta-CD, 2-hydroxylpropyl beta-CD, and gamma-CD scarcely induced the CF leakage from above the three liposomes. From the profiles of % CF leakage, together with measurements of differential scanning calorimetry, it was found that hydrophobic DOM-beta-CD penetrates the matrix of the liposomes to interact with them as well as TOM-beta-CD, and that less hydrophobic alpha-CD exists at the surface of the membrane to interact with the liposomes. Further, it was found that the interaction of CDs with liposomes changes depending not only on the length of fatty acid chain of phospholipid (condensation force and hydrophobicity) but also the hydrophobicity and the cavity size of CD.     

Pentaerythritol as the core of multivalent glycolipids: synthesis of a glycolipid with three SO3Lea ligands

A glycolipid containing three SO(3)Le(a) ligands was synthesized with pentaerythritol as the core. The glycolipid was used to prepare glycoliposomes that showed stability similar to that of DSPC liposomes without glycolipid. The easily prepared derivatives of pentaerythritol proved to be useful scaffolds for multivalent displaying of carbohydrates in the form of glycolipids and clustered glycoliposomes. [structure: see text]     

Investigation of Factors Affecting Controlled Release from Photosensitive DMPC and DSPC Liposomes

An investigation of liposomes comprised of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) lipids with cholesterol and zinc phthalocyanine (ZnPC) revealed that several fundamental liposome properties are influenced by composition and by lipid-specific features. DMPC and DSPC liposomes were synthesized, and their compositional changes, encapsulation capacities, morphologies, and release properties were evaluated. In this research, liposome degradation, lysis, and content release were initiated by photolysis, i.e., rupture induced by exposure to light. A controlled release mechanism was created through the introduction of photosensitizers (i.e., ZnPC) embedded within the cholesterol-stabilized liposome membrane. The light wavelength and light exposure time accelerated photodegradation properties of DMPC liposomes compared to DSPC liposomes, which exhibited a slower release rate. Morphological changes in the liposomes were strongly influenced by light wavelength and light exposure time. For both the DMPC and DSPC liposomes, visible light with wavelengths in the red end of the spectrum and broad spectrum ambient lighting (400?C700?nm) were more effective for lysis than UV-A light (365?nm). Heating liposomes to 100?°C decreased the stability of liposomes compared to liposomes kept at room temperatures. In addition, the optimal lipid-to-cholesterol-to-photoactivator ratio that produced the most stable liposomes was determined.     

Microfluidic directed formation of liposomes of controlled size

A new method to tailor liposome size and size distribution in a microfluidic format is presented. Liposomes are spherical structures formed from lipid bilayers that are from tens of nanometers to several micrometers in diameter. Liposome size and size distribution are tailored for a particular application and are inherently important for in vivo applications such as drug delivery and transfection across nuclear membranes in gene therapy. Traditional laboratory methods for liposome preparation require postprocessing steps, such as sonication or membrane extrusion, to yield formulations of appropriate size. Here we describe a method to engineer liposomes of a particular size and size distribution by changing the flow conditions in a microfluidic channel, obviating the need for postprocessing. A stream of lipids dissolved in alcohol is hydrodynamically focused between two sheathed aqueous streams in a microfluidic channel. The laminar flow in the microchannel enables controlled diffusive mixing at the two liquid interfaces where the lipids self-assemble into vesicles. The liposomes formed by this self-assembly process are characterized using asymmetric flow field-flow fractionation combined with quasi-elastic light scattering and multiangle laser-light scattering. We observe that the vesicle size and size distribution are tunable over a mean diameter from 50 to 150 nm by adjusting the ratio of the alcohol-to-aqueous volumetric flow rate. We also observe that liposome formation depends more strongly on the focused alcohol stream width and its diffusive mixing with the aqueous stream than on the sheer forces at the solvent-buffer interface.     

Resistive-pulse detection of multilamellar liposomes

The resistive-pulse method was used to monitor the pressure-driven translocation of multilamellar liposomes with radii between 190 and 450 nm through a single conical nanopore embedded in a glass membrane. Liposomes (0% and 5% 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (sodium salt) in 1,2-dilauroyl-sn-glycero-3-phosphocholine or 0%, 5%, and 9% 1,2-dipalmitoyl-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine) were prepared by extrusion through a polycarbonate membrane. Liposome translocation through a glass nanopore was studied as a function of nanopore size and the temperature relative to the lipid bilayer transition temperature, T(c). All translocation events through pores larger than the liposome, regardless of temperature, show translocation times between 30 and 300 μs and current pulse heights between 0.2% and 15% from the open pore baseline. However, liposomes at temperatures below the T(c) were captured at the pore orifice when translocation was attempted through pores of smaller dimensions, but squeezed through the same pores when the temperature was raised above T(c). The results provide insights into the deformation and translocation of individual liposomes through a porous material.     

Integrity of liposomes in presence of various formulation excipients, when dispersed in aqueous media and in hydrogels

The integrity of liposomes when dispersed in presence of various common formulation excipients is studied. Additionally, the effect of the excipients on the release of calcein from the same liposomes when dispersed in hydrogels is investigated and the results of the two sets of experiments are compared. Propyleneglycol (PG), transcutol CG (TR), cremophor EL (CR) and labrafac hydro WL 1219 (LB) are used at 10 or 25% (v/v) and the retention of liposome encapsulated calcein is followed for 24 or 48 h periods. Calcein entrapping multilamellar liposomes composed of phosphatidylcholine (PC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) with or without addition of different amounts of cholesterol (Chol) were prepared by the thin film hydration method.

Experimental results reveal that liposomes are affected more by the excipients in the order: LB > CR > PG  TR. Particularly LB and in some cases also CR result in rapid release of most or the entire vesicle encapsulated dye. Addition of Chol in both PC and DSPC liposomes results in substantial increase of vesicle integrity in all cases. Concerning the release of calcein form the liposomal gels, from DSPC/Chol (1:1) liposomal gels calcein release was not affected by addition of 25% of TR or PG in all gels studied, but LB caused a significant increase in calcein release. However, from PC-liposomal gels even TR and PG (at 25%), increases calcein release.

Conclusively, the results of this study suggest that liposomes are protected from excipients when dispersed in gels compared to aqueous media. This should be taken into account when liposomal drug formulations are designed.     

Physicochemical properties of PEG-grafted liposomes

Egg phosphatidylcholine (EggPC) or dimyristoylphosphatidylcholine (DMPC) liposomes containing polyethylene glycol (PEG)-lipids covering a range of 0-30 mol% have been prepared by Extrusion method. The physicochemical properties including size evolution and calcein permeation were evaluated to investigate the effect of PEG-lipids on bilayer structure. The results from quasielasetic light scattering (QELS), freeze-fracture microscopy, and gel exclusion chromatography revealed that presence of low concentration of PEG-lipid results in decreasing of vesicle size and further increase in the PEG-lipid concentrations lead to a transition from the lamellar membranes to micelles. The permeability for calcein increased with increase in concentration of distearoylphosphatidylethanolamine (DSPE)-PEG. On the other hand, the permeability decreased with low amount of cholesterol-PEG (blow 20% cholesterol-PEG) and increased with high amount of it. The maximum concentration of PEG-lipid that may be incorporated without alteration of the liposome structure depends on the composition of the bilayer. The concentration of DSPE-PEG2000 incorporated into vesicles without damaging vesicle structures were <20 mol% for EggPC and <10% for DMPC.     

Structure and properties of complexes of polycationic brushes with anionic liposomes

The adsorption of anionic lipid vesicles (liposomes) on the surfaces of colloidal particles containing grafted polycationic chains (cationic brushes) is studied. The stability of liposome-brush complexes in aqueous salt solutions increases with the content of anionic lipid in the liposomal membrane; complexes with liposomes containing 20 and 30 mol% anionic lipids do not dissociate into individual components in a 1.2 M NaCl solution. The integrity of the brush-bound liposomes is preserved. The developed approach can be used to obtain nanosized carriers for biologically active compounds.     

Dynamic and structural aspects of PEGylated liposomes monitored by NMR

Proton-detected NMR diffusion and (31)P NMR chemical shifts/bandwidths measurements were used to investigate a series of liposomal formulations where size and PEGylation extent need to be controlled for ultrasound mediated drug release. The width of the (31)P line is sensitive to aggregate size and shape and self-diffusion (1)H NMR conveys information about diffusional motion, size, and PEGylation extent. Measurements were performed on the formulations at their original pH, osmolality, and lipid concentration. These contained variable amounts of PEGylated phospholipid (herein referred to as PEG-lipid) and cholesterol. At high levels of PEG-lipid (11.5 and 15 mol%) the self-diffusion (1)H NMR revealed the coexistence of two entities with distinct diffusion coefficients: micelles (1.3 to 3x10(-11) m(2)/s) and liposomes (approximately 5x10(-12) m(2)/s). The (31)P spectra showed a broad liposome signal and two distinct narrow lines that were unaffected by temperature. The narrow lines arise from mixed micelles comprising both PEG-lipids and phospholipids. The echo decay in the diffusion experiments could be described as a sum of exponentials revealing that the exchange of PEG-lipid between liposomes and micellar aggregates is slower than the experimental observation time. For low amounts of PEG-lipid (1 and 4.5 mol%) the (31)P spectra consisted of a broad signal typically obtained for liposomes and the diffusion data were best described by a single exponential decay attributed solely to liposomes. For intermediate amounts of PEG-lipid (8 mol%), micellization started to occur and the diffusion data could no longer be fitted to a single or bi-exponential decay. Instead, the data were best described by a log-normal distribution of diffusion coefficients. The most efficient PEG-lipid incorporation in liposomes (about 8 mol%) was achieved for lower molecular weight PEG (2000 Da vs 5000 Da) and when the PEG-lipid acyl chain length matched the acyl chain length of the liposomal core phospholipid. Simultaneously to the PEGylation extent, self-diffusion (1)H NMR provides information about the size of micelles and liposomes. The size of the micellar aggregates decreased as the PEG-lipid content was increased while the liposome size remained invariant.     

Comparison of liposomes formed by sonication and extrusion: rotational and translational diffusion of an embedded chromophore

We report on the physical and optical characterization of liposomes formed by extrusion and sonication, two widely used methods for vesicle preparation. We also address the issue of whether the properties of bilayers formed from liposomes prepared by the two techniques differ at the molecular and mesoscopic levels. We used the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), with and without cholesterol, to form liposomes, incorporating 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (18:1-12:0 NBD-PC) as an optical probe of dynamics. We measured the physical morphology of liposomes by transmission electron microscopy (TEM) and dynamic light scattering (DLS), and the rotational and translational diffusion of 18:1-12:0 NBD-PC by time correlated single photon counting (TCSPC) and fluorescence recovery after pattern photobleaching (FRAPP), respectively. We find that, despite apparent differences in average size and size distribution, both methods of preparation produced liposomes that exhibit the same molecular scale environment. The translational diffusion behavior of the tethered chromophore in planar bilayer lipid membranes formed from the two types of liposomes also yielded similar results.     

The Role of Lateral Tension in Calcium-Induced DPPS Vesicle Rupture

We assess the role of lateral tension in rupturing anionic dipalmitoylphosphatidyserine (DPPS), neutral dipalmitoylphosphatidylcholine (DPPC), and mixed DPPS-DPPC vesicles. Binding of Ca(2+) is known to have a significant impact on the effective size of DPPS lipids and little effect on the size of DPPC lipids in bilayer structures. In the present work we utilized laser transmission spectroscopy (LTS) to assess the effect of Ca(2+)-induced stress on the stability of the DPPS and DPPC vesicles. The high sensitivity and resolution of LTS has permitted the determination of the size and shape of liposomes in solution. The results indicate a critical size after which DPPS single shell vesicles are no longer stable. Our measurements indicate Ca(2+) promotes bilayer fusion up to a maximum diameter of ca. 320 nm. These observations are consistent with a straightforward free-energy-based model of vesicle rupture involving lateral tension between lipids regulated by the binding of Ca(2+). Our results support a critical role of lateral interactions within lipid bilayers for controlling such processes as the formation of supported bilayer membranes and pore formation in vesicle fusion. Using this free energy model we are able to infer a lower bound for the area dilation modulus for DPPS (252 pN/nm) and demonstrate a substantial free energy increase associated with vesicle rupture.     

Kinetics of the adhesion of DMPC liposomes on a mercury electrode. Effect of lamellarity, phase composition, size and curvature of liposomes, and presence of the pore forming peptide mastoparan X

Liposomes suspended in aqueous electrolyte solutions can adhere at mercury electrodes. The adhesion is a complex process that starts with the docking and opening and leads to a spreading, finally resulting in the formation of islands of adsorbed lecithin molecules. The adhesion process can be followed by chronoamperometry, and a detailed analysis of the macroscopic and microscopic kinetics can be performed yielding rate constants and activation parameters. By using giant unilamellar liposomes and multilamellar liposomes, the effect of lamellarity and liposome size could be elucidated for liposomes in the liquid crystalline, gel, and superlattice phase states. Below the phase transition temperature, the time constant of opening of the liposomes (i.e., the irreversible binding of the lecithin molecules on the preliminary contact interface liposome|mercury and the therewith associated disintegration of the liposome membrane on that spot) is shown to be strongly size dependent. The activation energy, however, of that process is size independent with the exception of very small liposomes. That size dependence of time constants is a result of the size dependence of the initial contact area. The time constant and the activation energies of the spreading step exhibit a strong size dependence, which could be shown to be due to the size dependence of rate and activation energy of pore formation. Pore formation is necessary to release the solution included in the liposomes. This understanding was corroborated by addition of the pore inducing peptide Mastoparan X to the liposome suspension. The obtained results show that electrochemical studies of liposome adhesion on mercury electrodes can be used as a biomimetic tool to understand the effect of membrane properties on vesicle fusion.     

Influence of temperature on the colloidal stability of the F-DPPC and DPPC liposomes induced by lanthanum ions

The influence of La(3+) on the colloidal stability of liposomes made up by two zwitterionic phospholipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-[16-fluoropalmitoyl-phosphatidylcholine (F-DPPC), in aqueous media has been investigated by dynamic light scattering and electrophoretic mobility. The critical aggregation concentration (c.a.c.) of La(3+) for F-DPPC and DPPC liposomes were experimentally obtained, and the results were compared with theoretical predictions using the Derjaguin-Landau-Verwey-Overbeek theory. In order to evaluate the influence of the state of the bilayer on the stability of liposomes, all experiments were performed at temperatures below and above the chain-melting phase-transition temperature of lipids (transition temperature of lipids). Changes in the size of both types of liposomes and high values of polydispersity in the presence of La(3+) showed that these ions induce aggregation of liposomes at 25 °C and at 60 °C. At 25 °C, when the bilayer of F-DPPC liposomes is interdigited, DPPC liposomes are more resistant to aggregation than the liposomes formed with F-DPPC. However, this difference disappears at 60 °C, when both bilayers have the same conformation. The experimental results also indicate that the c.a.c. is higher at 60 °C than at 25 °C for both types of liposomes. In fact, it has been observed by dynamic light scattering measurements that aggregation of liposomes at 25 °C can be prevented by increasing the solution temperature for La(3+) concentrations near to the c.a.c. Moreover, the behavior of these liposomes in the presence of the ion was studied at temperatures above and below the transition temperature of the phospholipids.     

Novel asparagine-derived lipid enhances distearoylphosphatidylcholine bilayer resistance to acidic conditions

A novel asparagine-derived lipid analogue (ALA(11,17)) bearing a tetrahydropyrimidinone headgroup and two fatty chains (11 and 17 indicate the lengths of linear alkyl groups) was synthesized in high yield and purity. The thin film hydration of formulations containing 5 mol % or greater ALA(11,17) in distearoylphosphatidylcholine (DSPC) generated multilamellar vesicles (MLVs) that remained unaggregated according to optical microscopy, while those formed from DSPC only were highly clustered. The MLVs were processed into unilamellar liposomes via extrusion and were characterized by dynamic light scattering (DLS), zeta potential, turbidity, and scanning electron microscopy (SEM) analysis. Results show that the presence of ALA(11,17) in DSPC liposomes significantly alters the morphology, colloidal stability, and retention of encapsulated materials in both acidic and neutral conditions. The ability of ALA(11,17)-hybrid liposomes to encapsulate and retain inclusions under neutral and acidic conditions (pH < 2) was demonstrated by calcein dequenching experiments. DLS and SEM confirmed that ALA(11,17)/DSPC liposomes remained intact under these conditions. The bilayer integrity observed under neutral and acidic conditions and the likely biocompatibility of these fatty amino acid analogues suggest that ALA(11,17) is a promising additive for modulating phosphatidylcholine lipid bilayer properties.     

Structure of small actin-containing liposomes probed by atomic force microscopy: effect of actin concentration & liposome size

Actin-containing liposomes were prepared via extrusion through 400 and 600 nm pore diameter membranes at different monomeric actin concentrations in low ionic strength buffer (G-buffer). After subjecting the liposome dispersions to high ionic strength polymerization buffer (F-buffer), topological changes in liposome structure were studied using atomic force microscopy (AFM). Paired dumbbell, horseshoelike, and disklike assemblies were observed for actin-containing liposomes extruded through 400 and 600 nm pore diameter membranes. The topology of actin-containing liposomes was found to be highly dependent on both liposome size and actin concentration. At 1 mg/mL actin, the actin-containing liposomes transformed into a disklike shape, whereas, at 5 mg/mL actin, the actin-containing liposomes retained a spherical shape. On the basis of these observations, we hypothesize that actin could either polymerize on the surface of the inner leaflet of the liposome membrane or polymerize in the aqueous core of the liposome. We explain the associated shape changes induced in actin-containing liposomes on the basis of the hypothesized mechanism of actin polymerization inside the liposomes. At higher actin concentrations (5 mg/mL), we observed membrane-induced actin self-assembly in G-buffer, which implies that G-actin is able to interact directly with lipid bilayers at sufficiently high concentrations.     

Liposome formation of selectively-polymerized diene-containing phospholipids and their postpolymerization

Small and large unilamellar liposomes composed of 1,2-bis(2,4-octadecadienoyl)-sn-glycero-3-phosphorylcholine (DODPC) are prepared by sonication and extrusion, respectively. They are polymerized with water-insoluble radical initiator, azobis(isobutyronitrile) (AIBN) which can selectively polymerize diene groups in 1-acyl chains of the lipids. Polymerized liposomes are freeze-dried to obtain the polymerized liposome powder. There are two methods to redisperse lyophilized liposomes into water. The extrusion is an effective method to disperse them because the energy at extrusion is necessary only for redispersion, whereas the excess energy at sonication gives damage on liposome structure. There is no difference in stability between polymerized liposomes before and after redispersion with extrusion. DODPC polymers, obtained from free radical-initiated polymerization with AIBN, are linear and have polymerizable diene groups in 2-acyl chains. The liposome powder is therefore soluble in organic solvents. Reconstruction of polymerized liposomes is performed with lipid polymers having low or high molecular weight. The lipid polymers having high molecular weight provide stable large unilamellar liposomes by ethanol injection, but unstable small unilamellar liposomes are formed by sonication. The liposomes reconstructed from lipid polymers having low molecular weight are unstable regardless of their size. After reconstruction of liposomes selectively polymerized by AIBN, diene groups in 2-acyl chains are polymerized by water-soluble radical initiator or UV-irradiation to yield highly crosslinked structure. Their stability is improved remarkably by this postpolymerization.     

Physico-chemical properties and cytotoxicity assessment of PEG-modified liposomes containing human hemoglobin

PEGylated liposomes encapsulating human hemoglobin as oxygen carriers were prepared from purified carbonylhemoglobin (HbCO) solution and a lipid mixture composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol) 2000] (DMPE-PEG(2000)) and palmitic acid. Hemoglobin was extracted and purified from human blood samples. SDS-PAGE was used to assess its purity. Diameter of liposomes containing hemoglobin was controlled to approximately 200 nm using extrusion as measured by dynamic light scattering and transmission electron microscopy. Liposome size distributions were shown to remain unimodal over 14 days, even at different storage temperatures. Zeta potential measurements revealed that liposome containing hemoglobin have a net surface charge of -7.16+/-0.33 mV. Also, hemoglobin encapsulated in liposomes was able to perform several cycles of oxygen loading and unloading using oxygen (O(2)) and carbon monoxide (CO). The hemoglobin vesicle dispersion showed some toxicity as revealed by three in vitro assays in which endothelial cell (HUVECs) monolayers were exposed to these dispersions. Cytotoxicity was function of the liposome concentration in the culture medium.