Phospholipid-Block Copolymer Hybrid Vesicles with Lysosomal Escape Ability.

The success of nanoparticulate formulations in drug delivery depends on various aspects including their toxicity, internalization, and intracellular location. Vesicular assemblies consisting of phospholipids and amphiphilic block copolymers are an emerging platform, which combines the benefits from liposomes and polymersomes while overcoming their challenges. We report the synthesis of poly(cholesteryl methacrylate)- block-poly(2-(dimethylamino) ethyl methacrylate) (pCMA- b-pDMAEMA) block copolymers and their assembly with phospholipids into hybrid vesicles. Their geometry, their ζ-potential, and their ability to adsorb onto polymer-coated surfaces were assessed. Giant unilamellar vesicles were employed to confirm the presence of both the phospholipids and the block copolymer in the same membrane. Furthermore, the cytotoxicity of selected hybrid vesicles was determined in RAW 264.7 mouse macrophages, primary rat Kupffer cells, and human macrophages. The internalization and lysosomal escape ability of the hybrid vesicles were confirmed using RAW 264.7 mouse macrophages. Taken together, our findings illustrate that the reported hybrid vesicles are a promising complementary drug delivery platform for existing liposomes and polymersomes.

Novel catanionic surfactant vesicle vaccines protect against Francisella tularensis LVS and confer significant partial protection against F. tularensis Schu S4 strain.

Francisella tularensis is a Gram-negative immune-evasive coccobacillus that causes tularemia in humans and animals. A safe and efficacious vaccine that is protective against multiple F. tularensis strains has yet to be developed. In this study, we tested a novel vaccine approach using artificial pathogens, synthetic nanoparticles made from catanionic surfactant vesicles that are functionalized by the incorporation of either F. tularensis type B live vaccine strain (F. tularensis LVS [LVS-V]) or F. tularensis type A Schu S4 strain (F. tularensis Schu S4 [Schu S4-V]) components. The immunization of C57BL/6 mice with "bare" vesicles, which did not express F. tularensis components, partially protected against F. tularensis LVS, presumably through activation of the innate immune response, and yet it failed to protect against the F. tularensis Schu S4 strain. In contrast, immunization with LVS-V fully protected mice against intraperitoneal (i.p.) F. tularensis LVS challenge, while immunization of mice with either LVS-V or Schu S4-V partially protected C57BL/6 mice against an intranasal (i.n.) F. tularensis Schu S4 challenge and significantly increased the mean time to death for nonsurvivors, particularly following the i.n. and heterologous (i.e., i.p./i.n.) routes of immunization. LVS-V immunization, but not immunization with empty vesicles, elicited high levels of IgG against nonlipopolysaccharide (non-LPS) epitopes that were increased after F. tularensis LVS challenge and significantly increased early cytokine production. Antisera from LVS-V-immunized mice conferred passive protection against challenge with F. tularensis LVS. Together, these data indicate that functionalized catanionic surfactant vesicles represent an important and novel tool for the development of a safe and effective F. tularensis subunit vaccine and may be applicable for use with other pathogens.

Calcein release behavior from liposomal bilayer; influence of physicochemical/mechanical/structural properties of lipids.

The design of the drug delivery depends upon different parameters. One of the most noticeable factors in design of the drug delivery is drug-release profile which determines the site of action, the concentration of the drug at the time of administration, the period of time that the drug must remain at a therapeutic concentration. To get a better understanding of drug release, large unilamellar liposomes containing calcein were prepared using 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-palmitoyl-sn-glycero-3-phosphocholine, and a mixture of them; calcein was chosen as a model of hydrophilic drug. The calcein permeability across liposomal membrane (with different compositions) was evaluated on the basis of the first-order kinetic by spectrofluorometer. Also, the effects of liposome composition/fluidity as well as the incubation temperature/pH were investigated. Furthermore, we simulated the digestion condition in the gastrointestinal tract in humans, to mimic human gastro-duodenal digestion to monitor calcein release during the course of the digestion process. In vitro digestion model ''pH stat'' was used to systematically examine the influence of pH/enzyme on phospholipid liposomes digestion under simulated gastro-duodenal digestion. The results revealed that calcein permeates across liposomal membrane without membrane disruption. The release rate of calcein from the liposomes depends on the number and fluidity of bilayers and its mechanical/physical properties such as permeability, bending elasticity. Chemo-structural properties of drugs like as partition coefficient (Log P), H-bonding, polar surface area (PSA) are also determinative parameter in release behavior. Finally, stimulated emission depletion (STED) microscopy was used to study calcein translocation through liposomal bilayers.

Ibuprofen transdermal ethosomal gel: characterization and efficiency in animal models.

A new ibuprofen transdermal nanosystem, designed by using an ethosomal carrier, was characterized and tested for its pharmacokinetic profile and therapeutic effects in animal models. It was found that the ethosomal nanosystem contains unilamellar vesicles with a normal size distribution of about 200 nm. The drug applied transdermally from the ethosomal gel was present in plasma for a longer period of time as compared to the oral administration and showed a high relative bioavailability. Ibuprofen plasma concentration reached a Cmax of 74.11 +/- 18.52 microg/ml 2 hours post application on rat skin. The ibuprofen ethosomal gel had an efficient antipyretic effect in fevered rats. Animal body temperature decreased to normal value gradually with duration of action of at least 12 hours compared to only 7 hours after the oral treatment. The transdermal ibuprofen gel also induced an analgesic effect 30 minutes following its application lasting for at least 6 hours. Biochemical and clinical hematological analysis results of the blood taken from animals in all experimental groups and those of skin histological examination show that ibuprofen ethosomal gel is safe and does not irritate the skin. Data obtained in this work suggest that the designed ethosomal ibuprofen gel could be further investigated in humans for its antipyretic effect.

Haemolytic activity of liposomes: effect of vesicle size, lipid concentration and polyethylene glycol-lipid or arsonolipid incorporation.

The haemolysis caused by various types of liposomes was measured after incubation of liposomes with human red blood cell (erythrocyte) suspension. Liposomes composed of phospholipids and containing or not arsonolipids (arsonoliposomes) were tested. In some cases liposomes that were coated with polyethylene glycol (MW 2000), which were formulated by including 8 mol% DSPE-PEG2000 in their lipid membrane, were used. Multilamellar vesicles were prepared by the thin film hydration technique (conventional liposomes) or by the one-step technique (arsonoliposomes). Sonicated vesicles were produced by probe sonication of the initial liposome preparations. Phospholipid concentration in the liposome dispersions were measured by the Stewart assay, and adjusted accordingly. Haemolysis was measured after incubating 100 microl of liposome dispersions with 900 microl of red blood cell suspension (blood) for 1 h. The results reveal that the haemolysis caused, when liposomes are incubated in blood at concentrations below 0.16 mg (lipid)/ml (blood), was minimum. Only in case of Pegylated arsonoliposomes, significant haemolysis percents were observed. At higher lipid concentrations, 0.38 or 0.6 mg/ml, the haemolysis caused by arsonoliposomes was substantially increased, even in the cases of non-Pegylated arsonoliposomes. In most cases, especially when arsonolipid-containing liposomes were evaluated, vesicle size also had considerable effect on vesicle-induced haemolysis. Nevertheless, at concentrations which are relevant with liposomal drug administration in humans, all formulations tested demonstrated negligible haemolysis.