Investigating bolalipids as solubilizing agents for poorly soluble drugs: Effects of alkyl chain length on solubilization and cytotoxicity.

Synthetic single-chain bolalipids with symmetrical headgroups have shown potential in various pharmaceutical applications, such as the stabilization of liposome bilayers. Despite their amphiphilic character, synthetic bolalipids have not yet been investigated for their suitability as solubilizing agents for poorly soluble drug compounds. In this study, three synthetic single-chain bolalipids with increasing alkyl chain lengths (C22, C24 and C26) were investigated. All three bolalipids were able to achieve an increased solubility of the model drug, mefenamic acid, by approximately 180% in a pH 7.4 buffer compared to only a 102-105% increase achieved by sodium dodecyl sulfate (SDS) or the non-ionic surfactant pegylated hydroxystearate (PEG-HS). Subsequently, interfacial activity of bolalipids and their ability to destabilize liposomal bilayers were investigated. The C22 bolalipid exhibited a consistently lower interfacial activity, which was consistent with its significantly lower cytotoxicity in the macrophage-like cell line, J774. A1, compared to C24 and C26 counterparts. The mean IC50 values of the bolalipids tested (0.035-0.093 mM) were approximately 4-100-fold lower than that of SDS (0.401 mM) or PEG-HS (0.922 mM), with the mechanism of toxicity linked to increased cell membrane permeability, as is expected for surfactants. In summary, evidence from this study shows that decreasing the length of the bolalipid alkyl linker from C26 to C22 resulted in a significantly decreased cytotoxicity with no loss in drug solubilization efficiency.

Enhanced optical imaging properties of lipid nanocapsules as vehicles for fluorescent conjugated polymers.

Conjugated polymer nanoparticles (CPNs) have emerged as highly photostable probes for optical and photoacoustic imaging. However, the aggregation of conjugated polymer (CP) molecules upon nanoparticle formation is associated with fluorescence quenching, poor yields and mutable particle sizes. This study investigated whether the CP encapsulation within the liquid midchain triglyceride (MCT) core of lipid nanocapsules (LNCs) may achieve reduced packing of CP chains leading to a stable system with enhanced optical features. The red- and near infrared-emitting CPs, CN-PPV and PCPDTBT, showed precipitation and aggregation-induced quenching with concentrations >~25 µg/mL in MCT alone. Despite this, CP encapsulation within LNCs abolished quenching at concentrations up to 1500 µg/mL. PCPDTBT-LNCs exhibited a quantum yield of 2.8% and a higher signal:background ratio in an optical imaging phantom compared to literature reports of PCPDTBT encapsulated in PEG-PLGA nanoparticles. In contrast, PCPDTBT-LNCs had slightly lower photoacoustic amplitudes than reported PEG-PLGA systems. CP-LNCs were also stable in size (32 ± 0.7 nm) and photoluminescence over 21 days at 4 °C, 25 °C and 37 °C. In summary, encapsulation of CP within the liquid core of lipid nanocapsules enhances the optical properties of fluorescent CP.

Effect of saponin on erythrocytes.

Saponins, naturally occurring glycosides and triterpene glycosides in plants, are considered useful in the prophylaxis and treatment of several disorders, including malignancy. The effect of these substances is partly attributable to induction of both apoptosis and necrosis. Saponin has previously been shown to trigger hemolysis. Erythrocytes may avoid hemolysis by entering programmed cell death or eryptosis, characterized by cell shrinkage and cell membrane scrambling, leading to phosphatidylserine exposure at the erythrocyte surface. Eryptosis is triggered by increase of cytosolic Ca(2+) activity ([Ca(2+)](i)). The present study explored, whether exposure of human erythrocytes to saponin modifies [Ca(2+)](i), ceramide formation, hemolysis, and eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, hemolysis from hemoglobin release, [Ca(2+)](i) from Fluo3-fluorescence, and ceramide utilizing specific antibodies. A 24 h exposure to saponin (15 µg/ml) resulted in a significant increase of annexin V binding and a significant stimulation of hemolysis. Saponin (15 µg/ml) further increased [Ca(2+)](i) and ceramide formation. Annexin V binding was significantly blunted but not abrogated in the nominal absence of extracellular Ca(2+). Saponin thus triggers cell membrane scrambling, an effect partially due to entry of extracellular Ca(2+) and ceramide formation.

Stimulation of erythrocyte cell membrane scrambling by mitotane.

UNLABELLED: background: Mitotane (1,1-dichloro-2-[o-chlorophenyl]-2-[p-chlorophenyl]ethane), a cytostatic drug used for the treatment of adrenocortical carcinomas, is effective by triggering tumor cell apoptosis. In analogy to apoptosis of nucleated cells, eryptosis is the suicidal death of erythrocytes, which is typically paralleled by cell shrinkage and breakdown of cell membrane phosphatidylserine asymmetry with subsequent phosphatidylserine exposure at the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca(2+) concentration ([Ca(2+)]i). The present study tested, whether treatment of human erythrocytes with mitotane is followed by eryptosis. METHODS: [Ca(2+)]i was estimated from Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, and hemolysis from hemoglobin release. RESULTS: Exposure to mitotane (≥ 5 µg/ml ≈ 16 µM) significantly increased [Ca(2+)]i, increased annexin V binding and triggered hemolysis, but did not significantly modify forward scatter. The effect on annexin V binding was significantly blunted in the absence of extracellular Ca(2+). Within 30 min Ca(2+) ionophore ionomycin (1 µM) decreased forward scatter, an effect virtually abolished in the presence of mitotane (15 µg/ml). CONCLUSIONS: Mitotane increases [Ca(2+)]i with subsequent phosphatidylserine translocation. By the same token mitotane inhibits Ca(2+) induced cell shrinkage.

Effect of nitazoxanide on erythrocytes.

Nitazoxanide, a drug effective against a variety of pathogens, triggers apoptosis and is thus considered to be employed against malignancy. Similar to nucleated cells, erythrocytes may undergo an apoptosis-like suicidal cell death or eryptosis. Hallmarks of eryptosis include cell shrinkage and phospholipid scrambling of the cell membrane with translocation of phosphatidylserine to the erythrocyte surface. Stimulators of eryptosis include increase in cytosolic Ca(2+) -activity ([Ca(2+) ]i ). The Ca(2+) -sensitivity of eryptosis is increased by ceramide. This study explored whether nitazoxanide triggers eryptosis. [Ca(2+) ]i was estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin-V-binding, ceramide abundance utilizing fluorescent antibodies and haemolysis from haemoglobin release. A 48-hr exposure to nitazoxanide (1-50 µg/ml) did not significantly modify [Ca(2+) ]i but significantly increased ceramide formation, decreased forward scatter (≥10 µg/ml), increased the percentage of annexin-V-binding erythrocytes (≥10 µg/ml) and, at higher concentrations (≥20 µg/ml), stimulated haemolysis. The stimulation of annexin-V-binding was significantly blunted in the absence of calcium. Nitazoxanide thus stimulates eryptosis, an effect in part due to ceramide formation.

Estramustine-induced suicidal erythrocyte death.

BACKGROUND: The nitrogen mustard derivative of estradiol-17ß-phosphate estramustine is used for the treatment of prostate cancer. Estramustine may trigger suicidal death of cancer cells. Side effects of estramustine include anemia. At least in theory, estramustine could cause anemia by stimulation of eryptosis, the suicidal death of erythrocytes. Hallmarks of eryptosis include cell shrinkage, increased cytosolic Ca2+ activity ([Ca2+]), ceramide formation and phosphatidylserine translocation to the outer leaflet of the cell membrane with phosphatidylserine exposure at the erythrocyte surface. Eryptosis is stimulated by increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored whether estramustine triggers eryptosis. METHODS: [Ca2+]i was estimated from Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, and hemolysis from hemoglobin release. RESULTS: A 24 h exposure to estramustine (≤ 100 µM) significantly increased [Ca2+]i, increased annexin V binding and increased hemoglobin release. The effect of estramustine on annexin V binding was significantly blunted by removal of extracellular Ca2+. CONCLUSIONS: Estramustine stimulates both, eryptosis and hemolysis. The estramustine induced translocation of phosphatidylserine to the cell surface is at least partially due to increase of cytosolic Ca2+ activity.

Effect of thioridazine on erythrocytes.

BACKGROUND: Thioridazine, a neuroleptic phenothiazine with antimicrobial efficacy is known to trigger anemia. At least in theory, the anemia could result from stimulation of suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and by phospholipid scrambling of the cell membrane with phosphatidylserine exposure at the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca²âº-concentration ([Ca²âº](i)) and activation of p38 kinase. The present study explored, whether thioridazine elicits eryptosis. METHODS: [Ca²âº](i) has been estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin-V-binding, and hemolysis from hemoglobin release. RESULTS: A 48 hours exposure to thioridazine was followed by a significant increase of [Ca²âº](i) (30 µM), decrease of forward scatter (30 µM), and increase of annexin-V-binding (≥12 µM). Nominal absence of extracellular Ca²âº and p38 kinase inhibitor SB203580 (2 µM) significantly blunted but did not abolish annexin-V-binding following thioridazine exposure. CONCLUSIONS: Thioridazine stimulates eryptosis, an effect in part due to entry of extracellular Ca²âº and activation of p38 kinase.