Preparation of asymmetric phospholipid vesicles for use as cell membrane models.

Freely suspended liposomes are widely used as model membranes for studying lipid-lipid and protein-lipid interactions. Liposomes prepared by conventional methods have chemically identical bilayer leaflets. By contrast, living cells actively maintain different lipid compositions in the two leaflets of the plasma membrane, resulting in asymmetric membrane properties that are critical for normal cell function. Here, we present a protocol for the preparation of unilamellar asymmetric phospholipid vesicles that better mimic biological membranes. Asymmetry is generated by methyl-ß-cyclodextrin-catalyzed exchange of the outer leaflet lipids between vesicle pools of differing lipid composition. Lipid destined for the outer leaflet of the asymmetric vesicles is provided by heavy-donor multilamellar vesicles containing a dense sucrose core. Donor lipid is exchanged into extruded unilamellar acceptor vesicles that lack the sucrose core, facilitating the post-exchange separation of the donor and acceptor pools by centrifugation because of differences in vesicle size and density. We present two complementary assays allowing quantification of each leaflet's lipid composition: the overall lipid composition is determined by gas chromatography-mass spectrometry, whereas the lipid distribution between the two leaflets is determined by NMR, using the lanthanide shift reagent Pr3+. The preparation protocol and the chromatographic assay can be applied to any type of phospholipid bilayer, whereas the NMR assay is specific to lipids with choline-containing headgroups, such as phosphatidylcholine and sphingomyelin. In ~12 h, the protocol can produce a large yield of asymmetric vesicles (up to 20 mg) suitable for a wide range of biophysical studies.

Salvianolic acid B and its liposomal formulations: anti-hyperalgesic activity in the treatment of neuropathic pain.

Salvianolic acid B (SalB) represents the most characteristic constituent of Salvia miltiorrhiza Bge. with a strong free radicals scavenger activity. This property may be useful in the treatment of some severe chronic diseases, where there is an imbalance of reactive oxygen species formation and where intracellular reactive oxygen and nitrogen species level can cause severe cell damage and even cell death. In particular, SalB can protect against the oxidative stress as well as the antioxidant superoxide dismutase and reduced activity of glutathione, important determinants of neuropathological and behavioural consequences in neuropathic pain. This is a chronic disease defined by the WHO as an untreatable illness because therapeutics are unsatisfactory in many cases and there is an urgent need to discover and develop novel active drugs. In the present work, SalB has been extracted and purified with an efficient and rapid method from the roots and rhizome of S. miltiorrhiza Bge. It was firstly submitted to pharmacological studies using the paw-pressure test, in an animal model of neuropathic pain where a peripheral mono neuropathy was produced by a chronic constriction injury of the sciatic nerve. SalB was effective against mechanical hyperalgesia when administered intraperitoneally at the dose of 100mg/kg, 15 min after administration. Due to the poor chemical stability and bioavailability of SalB, liposomes were developed as drug carriers for parental administration. SalB-loaded liposomes were characterised in terms of particle size, polydispersity index, encapsulation efficacy and morphology. According to the in vivo studies, encapsulation, especially into PEGylated liposomes, increased and prolonged the antihyperalgesic activity 30 min after i.p. administration and the effect was still significant at 45 min. Thus, PEGylated formulation ameliorated the performance of drug delaying, increasing and prolonging in time its antihyperalgesic effect.

A facile route for creating "reverse" vesicles: insights into "reverse" self-assembly in organic liquids.

Reverse vesicles are spherical containers in organic liquids (oils) consisting of an oily core surrounded by a reverse bilayer. They are the organic counterparts to vesicles in aqueous solution and could potentially find analogous uses in encapsulation and controlled release. However, few examples of robust reverse vesicles have been reported, and general guidelines for their formation do not exist. We present a new route for forming stable unilamellar reverse vesicles in nonpolar organic liquids, such as cyclohexane and n-hexane. The recipe involves mixing short- and long-chain lipids (lecithins) with a trace of a salt such as sodium chloride. The ratio of short- to long-chain lecithin controls the type and size of self-assembled structure. As this ratio is increased, a spontaneous transition from reverse micelles to reverse vesicles occurs. Small-angle neutron scattering (SANS) and transmission electron microscopy (TEM) confirm the presence of unilamellar vesicles in the corresponding solutions. Average vesicle diameters can be tuned from 60 to 250 nm depending on the sample composition.

Increase of the pharmacological and pharmacokinetic efficacy of negatively charged polypeptide recombinant hirudin in rats via parenteral route by association with cationic liposomes.

Two biodegradable cationic lipids, stearylamine and DC-Chol, were chosen to investigate the effect of cationic lipids on the in vitro and in vivo characteristics of hydrophilic proteins or peptides of low isoelectric point. Thrombin inhibitor recombinant hirudin variant-2 (rHV2) was selected as the model drug. The cationic lipids were found to achieve higher entrapment efficiency of rHV2 in liposomes than zwitterionic lipids. The positively charged liposomes became less positive and relatively stable in serum after loading rHV2. The cationic liposomes induced sustained release of rHV2 in the presence of plasma, significantly prolonged the antithrombotic efficacy and plasma level of rHV2 after intravenous injection in rats in comparison with neutral lipid liposomes, especially for stearylamine group. Both clotting times correlated well with plasma rHV2 levels. No serious adverse events were observed and physical state of rats was satisfactory for all the formulations. Electrostatic interaction between negative charge of rHV2 and cationic liposomes was confirmed and it might affect all the characteristics of rHV2 loaded cationic vehicles. The findings suggest that cationic liposomes may be a potential sustained-release delivery system for parenteral administration of hydrophilic proteins or peptides with low isoelectric point to prolong efficacy and improve bioavailability.