stearates and Hemolysis

Safer and efficacious Amphotericin B (AmB) nanoformulations can be designed by augmenting AmB in the monomeric or super-aggregated state, and restricting the aggregated state, by choosing the appropriate excipient, which can be facilitated by employing in-silico prediction as a tool. Excipients selected for the study included linear fatty acids from caprylic (C

Topics: Amphotericin B; Antifungal Agents; Carbon; Ethylene Oxide; Excipients; Hemolysis; Humans; Molecular Docking Simulation; Polyenes; Polyethylene Glycols; Stearates; Surface-Active Agents

The commercially available alpha-asarone injections (CA-ARE) were frequently found to cause severe anaphylactic reactions by the solubilizer contained in the formulation such as polysorbate 80 and propylene glycol. This study aimed to develop a new ARE injection using Kolliphor HS 15 as solubilizing agent (HS 15-ARE) by the dissolution method to resolve its poor solubility problem and reduce the anaphylaxis of CA-AREs caused by Polysorbate 80. The HS 15-ARE micelle showed a homogeneous round shape with the mean particle size of around 13.73 ± 0.02 nm, polydisperse index (PDI) of 0.19 ± 0.01 and solubilizing efficiency of 95.7% ± 2.4%. In vitro and in vivo studies showed that HS 15-ARE is a stable injection presenting the same pharmacokinetic profile with CA-ARE. Moreover, improved therapeutic effect was observed for HS 15-ARE in treating asthma compared to CA-ARE (p < 0.05) with no anaphylactic reactions observed. These results demonstrate that the new formulation of ARE (HS 15-ARE) has a great potential for replacing CA-AREs injections.

Topics: Allylbenzene Derivatives; Anaphylaxis; Animals; Anisoles; Anti-Asthmatic Agents; Asthma; Biological Availability; Cells, Cultured; Chemistry, Pharmaceutical; Disease Models, Animal; Drug Stability; Erythrocytes; Guinea Pigs; Hemolysis; Histamine; Injections, Intravenous; Male; Molecular Structure; Particle Size; Polyethylene Glycols; Rats, Wistar; Sheep; Solvents; Stearates; Surface Properties; Tissue Distribution

Trapping in the endosomes is currently believed to represent the main barrier for transfection. Peptides, which allow endosomal escape have been demonstrated to overcome this barrier, similarly to the entry of viruses. However, the design principles of such endosomolytic peptides remain unclear. We characterized three analogs derived from membrane disrupting antimicrobial peptides (AMP), viz. LL-37, melittin, and bombolitin V, with glutamic acid substituting for all basic residues. These analogs are pH-sensitive and cause negligible membrane permeabilization and insignificant cytotoxicity at pH7.4. However, at pH5.0, prevailing in endosomes, membrane binding and hemolysis of human erythrocytes become evident. We first condensed the emerald green fluorescent protein (emGFP) containing plasmid by protamine, yielding 115 nm diameter soluble nanoplexes. For coating of the nanoplex surface with a lipid bilayer we introduced a hydrophobic tether, stearyl-octa-arginine (SR8). The indicated peptides were dissolved in methanol and combined with lipid mixtures in chloroform, followed by drying at RT under a nitrogen flow. The dry residues were hydrated with nanoplexes in Hepes, pH7.4 yielding after a 30 min incubation at RT,rather monodisperse nanoparticles having an average diameter of 150-300 nm, measured by DLS and cryo-TEM. Studies with cell cultures showed the above peptides to yield expression levels comparable to those obtained using Lipofectamine 2000. However, unlike the polydisperse aggregates formed upon mixing Lipofectamine 2000 and plasmid, the procedure described yields soluble, and reasonably monodisperse nanoparticles, which can be expected to be suitable for gene delivery in vivo, using intravenous injection.

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Cathelicidins; Endosomes; Erythrocytes; Gene Expression; Glutamic Acid; Green Fluorescent Proteins; Hemolysis; Humans; Hydrogen-Ion Concentration; Lipids; Melitten; Mice; Molecular Sequence Data; Nanoparticles; NIH 3T3 Cells; Oligopeptides; Particle Size; Peptides; Protamines; Stearates; Transfection

Poly(glycerol adipate) (PGA)-based nanoparticles are promising carriers for drug delivery with a wide range of available structures. The biodegradable polymer with pendant free hydroxyl groups can be diversely functionalized. In this study, the toxicity of different Stearoyl-PGA nanoparticles with respect to erythrocytes and HepG2 cells was assessed. These cells are crucial test systems for intravenously injected biomedical particles.. For this work, a series of PGA polyesters with 0, 20, 50 and 65 mol% of converted hydroxyl groups was synthesized with stearic acid (PGABB, S20, S50, S65). Nanoparticles were prepared with these polymers by an optimized nanoprecipitation method. Physicochemical characterization was performed by photon correlation spectroscopy and zeta potential measurement. Cell compatibility was studied by a hemolysis assay with separated red blood cells as well as a QBlue viability test and a modified LDH cytotoxicity assay with HepG2 cells.. Different self-stabilizing nanoparticles with narrow size distributions in the range of 100-140 nm were prepared. All tested nanoparticle samples were nontoxic for HepG2 cells. In fact, increased metabolic activity and proliferation was observed after 24 h incubation with the Stearoyl-PGA particles. Apart from PGAS20, all samples did not show any hemolytic effect. Hemolysis of PGAS20 particles could be considerably decreased by adding Poloxamer 188 during the preparation process.

Topics: Cell Survival; Drug Carriers; Erythrocytes; Hemolysis; Hep G2 Cells; Humans; Nanoparticles; Particle Size; Polyesters; Stearates

PEG-12-acyloxystearates constitute a novel class of pharmaceutical solubilizers and are synthesized from polyethylene glycol and 12-hydroxystearic acid, which has been esterified with a second acyl chain. The hemolytic activity of these surfactants decreases drastically with increasing pendant acyloxy chain length, and surfactants with an acyloxy chain of 14 carbon atoms or more are essentially nonhemolytic. In this paper, the interactions of PEG-12-acyloxystearates (acyloxy chain lengths ranging from 8 to 16 carbon atoms) with phosphatidylcholine vesicles, used as a model system for erythrocyte membranes, were studied in search of an explanation for the large variations in hemolytic activity. Surfactant-induced alterations of membrane permeability were investigated by studying the leakage of vesicle-entrapped calcein. It was found that all of the surfactants within the series interact with the vesicle membranes and cause slow leakage at elevated surfactant concentrations, but with large variations in leakage kinetics. The initial leakage rate decreases rapidly with increasing pendant acyloxy chain length. After prolonged incubation, on the other hand, the leakage is not a simple function of acyloxy chain length. The effect of the surfactants on membrane integrity was also investigated by turbidity measurements and cryo-transmission electron microscopy. At a surfactant/lipid molar ratio of 0.4, the vesicle membranes are saturated with surfactant. When the surfactant/lipid molar ratio is further increased, the vesicle membranes are progressively solubilized into mixed micelles. The rate of this process decreases strongly with increasing acyloxy chain length. When comparing the results of the different experiments, it can be concluded that there is no membrane permeabilization below saturation of the vesicle membranes. The large variations in the kinetics suggest that several steps are involved in the mechanism of leakage induced by PEG-12-acyloxystearates and that their relative rates vary with acyloxy chain length. The slow kinetics may in part be explained by the low critical micelle concentrations (CMCs) exhibited by the surfactants. The CMCs were found to be in the range of 0.003-0.025 microM.

Topics: Hemolysis; Membranes, Artificial; Micelles; Phosphatidylcholines; Polyethylene Glycols; Stearates; Surface-Active Agents

A direct hemolytic activity, dependent on phospholipase A (PLA) activity, was located in the particulate subcellular fraction (P30) of Trichomonas vaginalis. We identified soluble direct and indirect hemolytic activities in the spent medium and soluble fraction (S30) of T. vaginalis strain GT-13. Spent medium showed the highest specific indirect hemolytic activity (SIHA) at pH 6.0 (91 indirect hemolytic units [HU]/mg/hr). Spent medium and P30, but not S30, showed direct hemolytic activity. PLA activity was protein dose dependent and time dependent. The highest PLA activity was observed at pH 6.0. All trichomonad preparations showed phospholipase A1 (PLA A1) and phospholipase A2 (PLA A2) activities. Indirect and direct hemolytic activity and PLA A1 and PLA A2 diminished at pH 6.0 and 8.0 with increasing concentrations of Rosenthal's inhibitor. The greatest effect was observed with 80 microM at pH 6.0 on the SIHA of S30 (83% reduction) and the lowest at pH 8.0, also on the SIHA of S30 (26% reduction). In conclusion, T. vaginalis contains particulate and soluble acidic, and alkaline direct and indirect hemolytic activities, which are partially dependent on alkaline or acidic PLA A1 and PLA A2 enzymes. These could be responsible for the contact-dependent and -independent hemolytic and cytolytic activities of T. vaginalis.

Topics: Animals; Culture Media, Conditioned; Female; Hemolysis; Humans; Hydrogen-Ion Concentration; Phospholipases A; Phospholipases A1; Phospholipases A2; Stearates; Trichomonas vaginalis

Three tertiary amine-based detergents with zero, one, or two hydroxyl groups at various positions in their head group were characterized for their ability to promote the cytosolic delivery of macromolecules. Critical micellar concentrations (CMC) and membrane-bound pKa values of the lipid constructs increased with increasing head group polarity, ranging from 1-5 microM and 5.9 to 6.3, respectively. Fluorescence resonance energy transfer (FRET) and calcein leakage experiments revealed that when the amine group is protonated introduction of -OH moieties to detergent head groups enhanced their ability to interact with and permeabilize anionic, endosome-mimicking vesicles. Different formulations of a diethanolamine-based lipid (DEL) were further evaluated for pH-dependent hemolytic activity and ability to promote cytosolic delivery of macromolecules in vitro. Intact liposomes containing DEL at its maximum limit of incorporation were less efficient than DEL-containing micelles in promoting hemoglobin leakage from human erythrocytes at acidic pH. In HeLa cells, DEL-containing detergent micelles facilitated efficient cytosolic release of endocytosed macromolecules such as fluorescein-labeled dextran of MW 10 kDa. This observation was further corroborated by a functional assay based on antisense-mediated up-regulation of enhanced green fluorescent protein (EGFP). Taken together, our findings emphasize the key role of polar head groups and micellar architecture of pH-sensitive detergents in mediating endosomal permeabilization and the efficient cytosolic delivery of macromolecules.

Topics: Cell Membrane Permeability; Cytosol; Detergents; Fluoresceins; Fluorescent Dyes; HeLa Cells; Hemolysis; Humans; Hydrogen-Ion Concentration; Liposomes; Micelles; Morpholines; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylethanolamines; Rhodamines; Stearates

Stearoyl-poly-N-vinylpyrrolidone (PVP-stear) of various molecular weights (M(n) = 1500-5500) self-assemble in aqueous medium. Particles prepared from PVP-stear were characterized in terms of shape and size distribution, and the mechanical stability of the particles was studied. The interaction of PVP-stear and its aggregates with blood components was investigated. Aggregates formed by the polymers with M(n) = 1500-3500 in the presence of human serum are stable. The direct lytic action of PVP-stear preparations was studied using sheep and human erythrocytes. The influence of PVP-stear aggregates on the activation of complement system both on classical and alternative pathways was examined. The aggregates prepared from PVP-stear of various molecular weights had no effect on the activation of the complement system.

Topics: Animals; Blood; Complement Activation; Erythrocytes; Hemolysis; Humans; Light; Micelles; Models, Biological; Molecular Weight; Particle Size; Polymers; Pyrrolidinones; Rabbits; Scattering, Radiation; Sheep, Domestic; Stearates

Trichomonad total extracts (TTE), or vesicular (P30) and soluble (530) subcellular fractions from 3 pathogenic Trichomonas vaginalis strains (GT-3. GT-13. and GT-15), lysed both human and Sprague-Dawley rat erythrocytes in a time- and dose-dependent manner. The entire hemolytic activity of TTE was located in P30, showing 2 peaks of maximum activity, one at pH 6.0 and another at pH 8.0. in the presence of 1 mM Ca2+. Hemolytic activity on rat erythrocytes was greater at pH 6.0 16.71 +/- 0.33 hemolytic units IHU]/mg/hr to 11.60 +/- 0.24 HU/mg/hr) than at pH 8.0 (3.81 +/- 0.30 HU/mg/hr to 5.75 +/- 0.65 HU/mg/hr). and it was greater than that on human red blood cells at pH 6.0 (2.67 +/- 0.19 HU/mg/hr to 4.08 +/- 0.15 HU/mg/hr) or pH 8.0 (2.24 +/- 0.0 9 HU/mg/hr to 2.81 +/- 0.06 HU/mg/hr). The alkaline and acidic hemolytic activity diminished (60-93% at pH 6.0 and 78-93% at pH 8.0) by the effect of 80 microM Rosenthal's inhibitor, which also inhibited 27-45% and 29-54% trichomonad alkaline and acidic phospholipase A activities, respectively. Vesicles, vacuoles, and hydrogenosomes were rich in P30. Trichomonas vaginalis has a hemolytic PLA, which could be involved in its cytopathogenic mechanism.

Topics: Animals; Calcium; Dose-Response Relationship, Drug; Erythrocytes; Hemolysis; Humans; Hydrogen-Ion Concentration; Phospholipases A; Rats; Stearates; Trichomonas vaginalis; Virulence

Brief shift of purified C5 and C6 at 0 degrees C to pH 6.4, followed by immediate neutralization, results in the generation of a factor, designated C(56)a, that lyses erythrocytes together with C7, C8, and C9. We compared C(56)a and C5b6 generated by an alternative-pathway convertase, with regard to their action on different target cells. We found tht C(56)a is similar to C5b6 in the following properties: 1) Together with C7, C(56)a forms a stable intermediate on either sheep or guinea pig erythrocytes. 2) Membrane-bound C3b, or A2C incorporated in the membrane, enhances lysis by C(56)a-9, as well as lysis by C5b6-9. We also found that the lysis of EC(56)a7 or EC5b67 intermediates by C8 and C9 depends on the species of the erythrocytes and the species of C8 and C9. Thus, lysis of sheep erythrocytes is more efficient with guinea pig C8 and C9 than with human C8 and C9. In the case of guinea pig erythrocytes, this relationship is reversed, i.e., these cells lyse more efficiently when human C8 and C9 are used. Enhancement of lysis by membrane-bound C3b or A2C does not abrogate this species incompatibility pattern.

Topics: Animals; Complement Activating Enzymes; Complement Activation; Complement C3; Complement C3-C5 Convertases; Complement C5; Complement C6; Complement C7; Complement C8; Complement C9; Complement System Proteins; Guinea Pigs; Hemolysis; Humans; Hydrogen-Ion Concentration; Sheep; Species Specificity; Stearates

Vesicles of a variety of types of phosphatidylcholine have been shown to be suitable donors of phosphatidylcholine to intact rat erythrocytes in the presence of a specific phosphatidylcholine exchange protein. Coincident with the progression of phosphatidylcholine exchange is the onset of hemolysis, occurring at degrees of exchange characteristic for the type of phosphatidylcholine employed. During exchange with the dimyristoyl, dipalmitoyl and distearoyl species, hemolysis starts when 27%, 25% and 22% of the native phosphatidylcholine is replaced by these disaturated species, respectively. In the case of dielaidoylglycerophosphocholine and 1-stearoyl-2-oleoylglycerophosphocholine hemolysis starts after the introduction of 30% and 28%, respectively. In contrast, the replacement of native erythrocyte phosphatidylcholine with egg phosphatidylcholine, and the dioleoyl species up to levels of 60% does not result in rapid hemolysis. Despite such functional consequences, overall contents of the individual phospholipids and cholesterol are normal. Accompanying the biochemical events are morphologic changes, including echinocyte an spherocyte formation. The structure-function implications and possible mechanisms of hemolysis are discussed.

Topics: Animals; Cattle; Dimyristoylphosphatidylcholine; Erythrocyte Membrane; Erythrocytes; Hemolysis; Liposomes; Microscopy, Electron, Scanning; Phosphatidylcholines; Pulmonary Surfactants; Rats; Stearates; Structure-Activity Relationship