Lysolipid containing liposomes for transendothelial drug delivery

BACKGROUND: Designing efficient 'vectors', to deliver therapeutics across endothelial barriers, in a controlled manner, remains one of the key goals of drug development. Recently, transcytosis of liposome encapsulated fluorescence marker calcein across a tight cell barrier was studied. The most efficient liposomes were found to be liposomes containing sufficient amount of alkyl phospholipid (APL) perifosine. APLs have similar structure as lysophosphatidyl choline (LPC), since APLs were synthesized as metabolically stable analogues of LPC, which increases endothelial permeability directly by inducing endothelial cell contraction, resulting in formation of gaps between endothelial cells. Since one of the unique properties of lysolipid, containing liposomal formulations is dynamic equilibrium of lysolipids, which are distributed among liposomes, micelles, and free form, such liposomes represent a reservoir of free lysolipids. On the other hand lysolipid containing liposomes also represent a reservoir of an encapsulated hydrophilic drug. PRESENTATION OF THE HYPOTHESIS: We hypothesize that free lysolipids, with highest concentration in vicinity of drug carrying liposomes, compromise endothelial integrity, primarily where concentrations of liposomes is the highest, in a similar manner as LPC, by formation of gaps between endothelial cells. Liposome encapsulated drug, which leaks from liposomes, due to liposome destabilization, caused by lysolipid depletion, can therefore be efficiently transported across the locally compromised endothelial barrier. TESTING THE HYPOTHESIS: This hypothesis could be verified: by measuring binding of perifosine and other lysolipids to albumin and to lysophospholipid receptor (LPL-R) group; formation of stress fibers and subsequent cell contraction; activation of RhoA, and endothelial barrier dysfunction; by a synthesis of other LPC analogues with high critical micellar concentration and measuring their effect on transendothelial permeability in presence and absence of albumin. IMPLICATIONS OF THE HYPOTHESIS: We propose that lysolipid containing liposomal formulations might be used as nonspecific transendothelial transport vector, since leakage of liposome encapsulated active drug occurs simultaneously with the release of the lysolipids. The concentration of the active drug is therefore expected to be the highest at the site of compromised endothelial barrier. By appropriate choice of the lysolipids an endothelial barrier would stay open only for a short time. Use of such liposomes would potentially maximize the delivery of the drug while limiting the passage of toxic substances and pathogens across the endothelial barrier. Combining lysolipid containing liposomes with superparamagnetic iron oxide nanoparticles or a targeting ligand might be required to efficiently localize drug delivery to a disease affected tissue and to avoid endothelial disruption over the entire body

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like oligomeric clusters of these yellow nanoparticles of narrow size distribution are synthesized under ambient conditions via two methods. The delay-time method controls the number of subunits in the oligoclusters by varying the time between the addition of HAuCl4 to alkaline solution and the subsequent addition of reducing agent, NaSCN. The yellow oligoclusters produced range in size from ~3 to ~25 nm. This size range can be further extended by an add-on method utilizing hydroxylated gold chloride (Na+[Au(OH4-x)Clx]-) to auto-catalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3 nm to 70 nm. The crude oligocluster preparations display narrow size distributions and do not require further fractionation for most purposes. The oligoclusters formed can be concentrated >300 fold without aggregation and the crude reaction mixtures remain stable for weeks without further processing. Because these oligomeric clusters can be concentrated before derivatization they allow expensive derivatizing agents to be used economically. In addition, we present two models by which predictions of particle size can be made with great accuracy

Ca 2+ switches the effect of PS-containing membranes on Factor Xa from activating to inhibiting: implications for initiation of blood coagulation

Calcium (Ca2+) plays a pivotal role in cellular and organismal physiology. The Ca2+ ion has an intermediate protein-binding affinity, thus it can serve as an on/off switch in regulation of different biochemical processes. The serum level of ionized Ca2+ is regulated with normal ionized Ca2+ being in the range from 1.10 to 1.29 mM. Hypocalcaemia (free Ca2+ < 1.1mM) in critically ill patients is commonly accompanied by hemostatic abnormalities, ranging from isolated thrombocytopenia to complex defects such as disseminated intravascular coagulation, commonly thought to be due to insufficient functioning of anticoagulation pathways. A small amount of Factor Xa (fXa) produced by Factor VIIa and exposed tissue factor is key to initiating blood coagulation by producing enough thrombin to induce later stages of coagulation. FXa must bind to phosphatidylserine (PS)-containing membranes to produce thrombin at a physiologically significant rate. In this work, we show that overall fXa activity on PS-containing membranes is sharply regulated by a “Ca2+ switch” centered at 1.16 mM, below which fXa is active and above which fXa forms inactive dimers on PS-exposing membranes. Our data lead to a mathematical model that predicts the variation of fXa activity as a function of both calcium and membrane concentrations. Because the critical Ca2+ concentration is at the lower end of the normal plasma ionized Ca2+ concentration range, we propose a new regulatory mechanism by which local Ca2+ concentration switches fXa from an intrinsically active form to a form requiring its cofactor (fVa) to achieve significant activity

Phosphatidylserine-Induced Factor Xa Dimerization and Binding to Factor Va Are Competing Processes in Solution

A soluble, short chain phosphatidylserine, 1,2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS), binds to discrete sites on FXa, FVa, and prothrombin to alter their conformations, to promote FXa dimerization (Kd ~ 14 nM), and to enhance both the catalytic activity of FXa and the cofactor activity of FVa. In the presence of calcium, C6PS binds to two sites on FXa, one in the epidermal growth factor like (EGF) domain and one in the catalytic domain; the latter interaction is sensitive to Na+ binding and probably represents a protein recognition site. Here we ask whether dimerization of FXa and its binding to FVa in the presence of C6PS are competitive processes. We monitored FXa activity at 5, 20 and 50 nM FXa while titrating with FVa in the presence of 400 µM C6PS and 3 or 5 mM Ca2+ to show that the apparent Kd of FVa-FXa interaction increased with increasing FXa concentration at 5 mM Ca2+, but the Kd was only slightly affected at 3 mM Ca2+. A mixture of 50 nM FXa and 50 nM FVa in the presence of 400 µM C6PS yielded both Xa homodimers and Xa ·Va heterodimers but no FXa dimers bound to FVa. A mutant FXa (R165A) that has reduced prothrombinase activity showed both reduced dimerization (Kd~147 nM) and reduced FVa binding (apparent Kd, = 58, 92 and 128 nM, respectively for 5, 20 and 50 nM R165A FXa). Native gel electrophoresis showed that the GLA-EGFNC fragment of FXa (lacking the catalytic domain) neither dimerized nor formed a complex with FVa in the presence of 400 µM C6PS and 5 mM Ca2+. Our results demonstrate that the dimerization site and FVa binding site are both located in the catalytic domain of FXa and that these sites are linked thermodynamically

Hemagglutinin Fusion Peptide Mutants in Model Membranes: Structural Properties, Membrane Physical Properties, and PEG-Mediated Fusion

While the importance of viral fusion peptides (e.g., hemagglutinin (HA) and gp41) in virus-cell membrane fusion is established, it is unclear how these peptides enhance membrane fusion, especially at low peptide/lipid ratios for which the peptides are not lytic. We assayed wild-type HA fusion peptide and two mutants, G1E and G13L, for their effects on the bilayer structure of 1,2-dioleoyl-3-sn-phosphatidylcholine/1,2-dioleoyl-3-sn-phosphatidylethanolamine/Sphingomyelin/Cholesterol (35:30:15:20) membranes, their structures in the lipid bilayer, and their effects on membrane fusion. All peptides bound to highly curved vesicles, but fusion was triggered only in the presence of poly(ethylene glycol). At low (1:200) peptide/lipid ratios, wild-type peptide enhanced remarkably the extent of content mixing and leakage along with the rate constants for these processes, and significantly enhanced the bilayer interior packing and filled the membrane free volume. The mutants caused no change in contents mixing or interior packing. Circular dichroism, polarized-attenuated total-internal-reflection Fourier-transform infrared spectroscopy measurements, and membrane perturbation measurements all conform to the inverted-V model for the structure of wild-type HA peptide. Similar measurements suggest that the G13L mutant adopts a less helical conformation in which the N-terminus moves closer to the bilayer interface, thus disrupting the V-structure. The G1E peptide barely perturbs the bilayer and may locate slightly above the interface. Fusion measurements suggest that the wild-type peptide promotes conversion of the stalk to an expanded trans-membrane contact intermediate through its ability to occupy hydrophobic space in a trans-membrane contact structure. While wild-type peptide increases the rate of initial intermediate and final pore formation, our results do not speak to the mechanisms for these effects, but they do leave open the possibility that it stabilizes the transition states for these events

Lysolipid containing liposomes for transendothelial drug delivery

Abstract Background Designing efficient 'vectors', to deliver therapeutics across endothelial barriers, in a controlled manner, remains one of the key goals of drug development. Recently, transcytosis of liposome encapsulated fluorescence marker calcein across a tight cell barrier was studied. The most efficient liposomes were found to be liposomes containing sufficient amount of alkyl phospholipid (APL) perifosine. APLs have similar structure as lysophosphatidyl choline (LPC), since APLs were synthesized as metabolically stable analogues of LPC, which increases endothelial permeability directly by inducing endothelial cell contraction, resulting in formation of gaps between endothelial cells. Since one of the unique properties of lysolipid, containing liposomal formulations is dynamic equilibrium of lysolipids, which are distributed among liposomes, micelles, and free form, such liposomes represent a reservoir of free lysolipids. On the other hand lysolipid containing liposomes also represent a reservoir of an encapsulated hydrophilic drug. Presentation of the hypothesis We hypothesize that free lysolipids, with highest concentration in vicinity of drug carrying liposomes, compromise endothelial integrity, primarily where concentrations of liposomes is the highest, in a similar manner as LPC, by formation of gaps between endothelial cells. Liposome encapsulated drug, which leaks from liposomes, due to liposome destabilization, caused by lysolipid depletion, can therefore be efficiently transported across the locally compromised endothelial barrier. Testing the hypothesis This hypothesis could be verified: by measuring binding of perifosine and other lysolipids to albumin and to lysophospholipid receptor (LPL-R) group; formation of stress fibers and subsequent cell contraction; activation of RhoA, and endothelial barrier dysfunction; by a synthesis of other LPC analogues with high critical micellar concentration and measuring their effect on transendothelial permeability in presence and absence of albumin. Implications of the hypothesis We propose that lysolipid containing liposomal formulations might be used as nonspecific transendothelial transport vector, since leakage of liposome encapsulated active drug occurs simultaneously with the release of the lysolipids. The concentration of the active drug is therefore expected to be the highest at the site of compromised endothelial barrier. By appropriate choice of the lysolipids an endothelial barrier would stay open only for a short time. Use of such liposomes would potentially maximize the delivery of the drug while limiting the passage of toxic substances and pathogens across the endothelial barrier. Combining lysolipid containing liposomes with superparamagnetic iron oxide nanoparticles or a targeting ligand might be required to efficiently localize drug delivery to a disease affected tissue and to avoid endothelial disruption over the entire body.</p

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

Reducing dilute aqueous HAuCl(4) with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like oligomeric clusters of these yellow nanoparticles of narrow size distribution are synthesized under ambient conditions via two methods. The delay-time method controls the number of subunits in the oligoclusters by varying the time between the addition of HAuCl(4) to alkaline solution and the subsequent addition of reducing agent, NaSCN. The yellow oligoclusters produced range in size from ~3 to ~25 nm. This size range can be further extended by an add-on method utilizing hydroxylated gold chloride (Na(+)[Au(OH(4-x))Cl(x)](-)) to auto-catalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3 nm to 70 nm. The crude oligocluster preparations display narrow size distributions and do not require further fractionation for most purposes. The oligoclusters formed can be concentrated >300 fold without aggregation and the crude reaction mixtures remain stable for weeks without further processing. Because these oligomeric clusters can be concentrated before derivatization they allow expensive derivatizing agents to be used economically. In addition, we present two models by which predictions of particle size can be made with great accuracy

Influence of cancerostatic perifosine on membrane fluidity of liposomes and different cell lines as measured by electron paramagnetic resonance

AIM: To test whether membrane fluidity and its changes are important for the sensitivity of cells to the action of perifosine (OPP), a new anticancer drug targeting cell membrane and not DNA. METHOD: Influence of OPP on the membrane structure of OPP-resistant MCF7, and OPP-sensitive MT3 breast cancer cell lines, as well as of mouse fibroblasts (L929) cell lines, and model cells (liposomes) was investigated by electron paramagnetic resonance, using spin labeled derivative of OPP (P5) and 5-doxylpalmitoyl methylester (MeFASL(10,3)) as spin probes. RESULTS: OPP increased membrane fluidity of all cell lines at concentrations higher than 50 µM (on the level of P ≤ 0.05, t test). In cells, the differences were observed only by P5 and not by MeFASL(10,3). Average order parameter S(eff) decreased for about 12% in MCF7 and L929 and only for 8% in OPP-sensitive MT3 cells, showing that there was no correlation between membrane fluidity changes and sensitivity of cells to OPP. The only correlation we found was between OPP sensitivity and the cell growth rate. In liposomes, both spin probes were sensitive to the action of OPP. S(eff) decreased with increasing concentration of OPP. For MeFASL(10,3) a significant decrease was observed at 4 mol% OPP, while for P5 it was observed at 8 mol%. CONCLUSION: Influence of OPP on plasma membrane fluidity of breast cancer cells is not the determining factor in the sensitivity of cells to OPP