Effect of fatty acids on the permeability barrier of model and biological membranes.

Because of the amphipathicity and conical molecular shape of fatty acids, they can efficiently incorporate into lipid membranes and disturb membrane integrity, chain packing, and lateral pressure profile. These phenomena affect both model membranes as well as biological membranes. We investigated the feasibility of exploiting fatty acids as permeability enhancers in drug delivery systems for enhancing drug release from liposomal carriers and drug uptake by target cells. Saturated fatty acids, with acyl chain length from C8 to C20, were tested using model drug delivery liposomes of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the breast cancer MCF-7 cell line as a model cell. A calcein release assay demonstrated reduction in the membrane permeability barrier of the DPPC liposomes, proportionally to the length of the fatty acid. Differential scanning calorimetry (DSC) and dynamic light scattering (DLS) experiments revealed that C12 to C20 fatty acids can stabilize DPPC liposomal bilayers and induce the formation of large structures, probably due to liposome aggregation and bilayer morphological changes. On the other hand, the short fatty acids C8 and C10 tend to destabilize the bilayers and only moderately cause the formation of large structures. The effect of fatty acids on DPPC liposomes was not completely transferrable to the MCF-7 cell line. Using cytotoxicity assays, the cells were found to be relatively insensitive to the fatty acids at apoptotic sub-millimolar concentrations. Increasing the fatty acid concentration to few millimolar substantially reduced the viability of the cells, most likely via the induction of necrosis and cell lysis. A bioluminescence living-cell-based luciferase assay showed that saturated fatty acids in sub-cytotoxic concentrations cannot reduce the permeability barrier of cell membranes. Our results confirm that the membrane perturbing effect of fatty acids on model membranes cannot simply be carried over to biological membranes of live cells.

Optimization and modeling of the remote loading of luciferin into liposomes.

We carried out a mechanistic study to characterize and optimize the remote loading of luciferin into preformed liposomes of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPC/DPPG) 7:3 mixtures. The influence of the loading agent (acetate, propionate, butyrate), the metal counterion (Na(+), K(+), Ca(+2), Mg(+2)), and the initial extra-liposomal amount of luciferin (nL(add)) on the luciferin Loading Efficiency (LE%) and luciferin-to-lipid weight ratio, i.e., Loading Capacity (LC), in the final formulation was determined. In addition, the effect of the loading process on the colloidal stability and phase behavior of the liposomes was monitored. Based on our experimental results, a theoretical model was developed to describe the course of luciferin remote loading. It was found that the highest luciferin loading was obtained with magnesium acetate. The use of longer aliphatic carboxylates or inorganic proton donors pronouncedly reduced luciferin loading, whereas the effect of the counterion was modest. The remote-loading process barely affected the colloidal stability and drug retention of the liposomes, albeit with moderate luciferin-induced membrane perturbations. The correlation between luciferin loading, expressed as LE% and LC, and nL(add) was established, and under our conditions the maximum LC was attained using an nL(add) of around 2.6µmol. Higher amounts of luciferin tend to pronouncedly perturb the liposome stability and luciferin retention. Our theoretical model furnishes a fair quantitative description of the correlation between nL(add) and luciferin loading, and a membrane permeability coefficient for uncharged luciferin of 1×10(-8)cm/s could be determined. We believe that our study will prove very useful to optimize the remote-loading strategies of moderately polar carboxylic acid drugs in general.

Enzymatic action of phospholipase A2 on liposomal drug delivery systems.

The overexpression of secretory phospholipase A2 (sPLA2) in tumors has opened new avenues for enzyme-triggered active unloading of liposomal antitumor drug carriers selectively at the target tumor. However, the effects of the liposome composition, drug encapsulation, and tumor microenvironment on the activity of sPLA2 are still not well understood. We carried out a physico-chemical study to characterize the sPLA2-assisted breakdown of liposomes using dye-release assays in the context of drug delivery and under physiologically relevant conditions. The influence of temperature, lipid concentration, enzyme concentration, and drug loading on the hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Tm=42°C) liposomes with snake venom sPLA2 was investigated. The sensitivity of human sPLA2 to the liposome composition was checked using binary lipid mixtures of phosphatidylcholine (PC) and phosphatidylglycerol (PG) phospholipids with C14 and C16 acyl chains. Increasing temperature (36-41°C) was found to mainly shorten the enzyme lag-time, whereas the effect on lipid hydrolysis rate was modest. The enzyme lag-time was also found to be inversely dependent on the lipid-to-enzyme ratio. Drug encapsulation can alter the hydrolysis profile of the carrier liposomes. The activity of human sPLA2 was highly sensitive to the phospholipid acyl-chain length and negative surface charge density of the liposomes. We believe our work will prove useful for the optimization of sPLA2-susceptible liposomal formulations as well as will provide a solid ground for predicting the hydrolysis profile of the liposomes in vivo at the target site.

Development of a cell-based bioassay for phospholipase A2-triggered liposomal drug release.

The feasibility of exploiting secretory phospholipase A2 (sPLA2) enzymes, which are overexpressed in tumors, to activate drug release from liposomes precisely at the tumor site has been demonstrated before. Although the efficacy of the developed formulations was evaluated using in vitro and in vivo models, the pattern of sPLA2-assisted drug release is unknown due to the lack of a suitable bio-relevant model. We report here on the development of a novel bioluminescence living-cell-based luciferase assay for the monitoring of sPLA2-triggered release of luciferin from liposomes. To this end, we engineered breast cancer cells to produce both luciferase and sPLA2 enzymes, where the latter is secreted to the extracellular medium. We report on setting up a robust and reproducible bioassay for testing sPLA2-sensitive, luciferin remote-loaded liposomal formulations, using 1,2-distearoyl-sn-glycero-3-phosphatidylcholine/1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPC/DSPG) 7:3 and DSPC/DSPG/cholesterol 4:3:3 as initial test systems. Upon their addition to the cells, the liposomes were degraded almost instantaneously by sPLA2 releasing the encapsulated luciferin, which provided readout from the luciferase-expressing cells. Cholesterol enhanced the integrity of the formulation without affecting its susceptibility to sPLA2. PEGylation of the liposomes only moderately broadened the release profile of luciferin. The provided bioassay represents a useful tool for monitoring active drug release in situ in real time as well as for testing and optimizing of sPLA2-sensitive lipid formulations. In addition, the bioassay will pave the way for future in-depth in vitro and in vivo studies.

Interaction of linear polyamines with negatively charged phospholipids: the effect of polyamine charge distance.

The binding of cationic polyamines to negatively charged lipid membranes is driven by electrostatic interactions and additional hydrophobic contributions. We investigated the effect of polyamines with different number of charges and charge separation on the phase transition behavior of vesicles of phosphatidylglycerols (dipalmitoylphosphatidylglycerol and dimyristoylphosphatidylglycerol) to differentiate between effects caused by the number of charges, the charge distance, and the hydrophobicity of the methylene spacer. Using differential scanning calorimetry and Fourier transform infrared spectroscopy complemented with monolayer experiments, we found that the binding constant of polyamines to negatively charged lipid vesicles depends as expected on the number of charges. However, for diamines, the effect of binding on the main phase transition of phosphatidylglycerols (PGs) is also strongly influenced by the charge distance between the ammonium groups in the backbone. Oligoamines with charges separated by two or three methylene groups bind more strongly and have larger stabilizing effects on the lipid gel phase of PGs. With multivalent polyamines, the appearance of several transition peaks points to effects of molecular crowding on the surface, i.e., binding of only two or three charges to the surface in the case of spermine, and possible concomitant domain formation.

The helical propensity of KLA amphipathic peptides enhances their binding to gel-state lipid membranes.

The role and importance of the conformation of antimicrobial peptides for their binding and incorporation into lipid membranes as well as for their bioactivity are still not well understood. In this paper, we studied the interaction between four cationic alpha-helical KLA peptides, which differ primarily in their helical propensity, and the anionic gel-state lipid DPPG (1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol). Of particular interest was the influence of the peptide conformation and membrane surface properties on the electrostatic binding process. Dynamic light scattering (DSL) showed that generally the KLA peptides possess high aggregation power but modest solubilization power. Circular dichroism spectroscopy (CD) spectra revealed that the KLA peptides with the low helical propensity tend to form beta-structures at low lipid/peptide ratios. Differential scanning calorimetry (DSC) thermograms showed that the helical KLA peptides stabilize the DPPG bilayer, whereas the beta-structured peptides induce pronounced membrane perturbations. Isothermal titration calorimetry (ITC) isotherms showed that the helical KLA peptides bind more efficiently to DPPG vesicles than the beta-structured KLA peptides, and that the binding affinity of the peptides is proportional to the peptide helical propensity and membrane negative surface charge. The stoichiometry values (N) deduced from the ITC isotherms suggest that the helical KLA peptides have a higher capacity to translocate the DPPG lipid bilayer. The new data presented in this study demonstrate the flexibility of KLA peptides in adopting various conformations in response to the surrounding and also how the peptide structuring controls the mode of peptide-membrane interaction.

Membrane-perturbing effect of fatty acids and lysolipids.

Due to their amphipathicity fatty acids and lysolipids incorporate into lipid membranes and may hence exert an effect on membrane permeability, morphology, and stability. Several studies have shown that fatty acids and lysolipids can reduce the permeability barrier of model membranes. The origin of this phenomenon may be related to changes in the curvature stress of the membrane caused by the effective non-cylindrical geometry of fatty acids and lysolipids. Therefore, it has been proposed that the same effects may carry over to apply to the permeability barrier of cell membranes, in which case the effect could possibly be exploited to enhance intracellular drug uptake. However, fatty acids and lysolipids are in themselves cytotoxic in micromolar concentrations. Experiments with living cells have shown that fatty acids and lysolipids at concentrations below their cytotoxicity limit cannot render cell membranes more permeable by perturbing the lipid bilayer component of the membrane. We summarize the limited, though, conclusive, available literature on this topic. The picture that emerges from this discussion illustrates the importance of a lipidology-based view for the rational development of liposomal drug-delivery systems. It is also an example of possible limitations in translating knowledge from simple lipid bilayers to real biological membranes.

Phospholipase A(2)-susceptible liposomes of anticancer double lipid-prodrugs.

A novel approach to anticancer drug delivery is presented based on lipid-like liposome-forming anticancer prodrugs that are susceptible to secretory phospholipase A(2) (sPLA(2)) that is overexpressed in several cancer types. The approach provides a selective unloading of anticancer drugs at the target tissues, as well as circumvents the necessity for "conventional" drug loading. In our attempts to improve the performance of the liposomes in vivo, several PEGylated and non-PEGylated liposomal formulations composed of a retinoid prodrug premixed with the sPLA(2)-hydrolyzable DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) were prepared. Besides favorably modifying the physicochemical properties of the liposomes, the incorporation of DPPC and PEG-lipids in the liposomes should substantially enhance the enzymatic activity, as concluded from literature. In addition, one can reap benefits from the presumed permeability enhancing effect of the liberated fatty acids and lysolipids. The size distribution of the prepared liposomes as well as their phase behavior, enzymatic hydrolysis, and cytotoxicity, in the presence and absence of sPLA(2), were determined. The liposomes were around 100nm in diameter and in the gel/fluid coexistence region at 37°C. The enzymatic hydrolysis of the prodrug was pronouncedly accelerated upon the premixing with DPPC, and the hydrolysis was further enhanced by PEGylation. Interestingly, the faster hydrolysis of the prodrug and the released fatty acids and lysolipids from DPPC did not improve the cytotoxicity of the mixture; the effect of combining the prodrug with DPPC was additive and not synergistic. The data presented here question the significance of the permeability enhancing effects claimed for fatty acids and lysolipids at the target cell membrane, and whether these effects can be achieved using physiologically achievable concentrations of fatty acids and lysolipids.

Characterization of fluorinated catansomes: a promising vector in drug-delivery.

Catansomes, which are vesicles prepared from mixtures of oppositely charged surfactants, have been suggested as effective alternatives to phospholipid vesicles, i.e., liposomes, in applications such as drug-delivery. This is mainly due to their enhanced chemical and physical stability as well as to their relatively easy preparation, which is an advantage for large-scale productions. In this study we have investigated catansomes prepared from a perfluorinated anionic surfactant (sodium perfluorooctanoate) premixed with a hydrogenated cationic surfactant (dodecyltrimethylammonium bromide or 1-dodecylpyridinium chloride). The aim was to gain insights into the physicochemical properties of these systems, such as size, stability, surface charge, and membrane morphology, which are essential for their use in drug-delivery applications. The catansomes were mostly unilamellar and 100-200 nm in size, and were stable for more than five months at room temperature. After loading the catansomes with the fluorescent marker calcein, they were found to exhibit an appreciable encapsulation efficiency and a low calcein leakage over time. The addition of fatty acids to calcein-loaded catansomes considerably promoted the release of calcein, and the rate and efficiency of calcein release were found to be proportional to the fatty acid concentration and chain length. Our results prove the feasibility of utilizing catansomes as drug-delivery vehicles as well as provide a means to efficiently release the encapsulated load.

Anticancer double lipid prodrugs: liposomal preparation and characterization.

The escape of encapsulated anticancer drugs from liposomes by passive diffusion often leads to suboptimal drug concentrations in the cancer tissue, therefore calling for effective trigger mechanisms to release the drug at the target. We investigated mixtures of lipid components that not only form stable liposomes, but also can be turned into active drugs by secretory phospholipase A2 (sPLA2), an enzyme that is upregulated in various cancer cells, without the necessity for conventional liposome drug loading. The liposomes are composed of a novel lipid-based retinoid prodrug premixed with saturated phospholipids. The prodrug is found to be miscible with phospholipids, and the lipid mixtures are shown to form liposomes with the desired size distribution. The preparation procedure, phase behavior, and physicochemical properties of the formed liposomes are described as a function of lipid composition. We show that the premixing of the prodrug with phospholipids can be used to modify the physicochemical properties of liposomal formulations. The results should prove useful for further exploration of the potential for using these novel lipid prodrugs in liposomal formulations for cancer treatment.

The binding of an amphipathic peptide to lipid monolayers at the air/water interface is modulated by the lipid headgroup structure.

We used monolayer techniques combined with infrared reflection absorption spectroscopy (IRRAS) to study the behavior of the 18-mer cationic peptide KLA1 (KLAL KLAL KAW KAAL KLA-NH2) at the air/water interface as well as its interaction with lipid films of different composition. The adsorption of the peptide from the subphase to the air/water interface was observed measuring the increase in surface pressure (π) at constant surface area. The binding of the peptide to lipid monolayers was followed by recording the change in lipid area at a constant surface pressure (π = 30 mN m(-1)). At the air/water interface, the peptide initially adopted an α-helix at large surface area per molecule, that is, low surface pressure, but further accumulation of the peptide at the interface induced a conformational change from α-helix to intermolecular ß-sheet, driven by intermolecular aggregation. When the peptide was injected into the subphase underneath lipid monolayers, it adsorbed pronouncedly to anionic monolayers containing phosphatidylglycerol forming an α-helix, but not to zwitterionic lipid monolayers. The large change in area observed upon peptide binding suggests that the peptide helix was incorporated into the apolar chain region of the lipids. An apparent partition coefficient of (0.3-1) × 10(6) M(-1) could be calculated for binding to pure POPG monolayers. Significant differences in binding affinity were observed comparing PG/PC with PG/PE monolayers, with the latter showing a higher binding constant. This shows that not only electrostatic and hydrophobic effects but also specific interactions between the headgroups of the lipids and the peptide side chains modulate the binding affinity.

Morphological changes induced by the action of antimicrobial peptides on supported lipid bilayers.

We utilized epifluorescence microscopy to investigate the morphological changes in labeled lipid bilayers supported on quartz surfaces (SLBs) induced by the interaction of cationic antimicrobial peptides with the lipid membranes. The SLBs were prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and mixtures thereof as well as from Escherichia coli lipid extract. We succeeded in the preparation of POPG and POPG-rich SLBs without the necessity to use fusogenic agents such as calcium by using the Langmuir-Blodgett/Langmuir-Schaefer transfer method. The adsorption of the peptides to the SLBs was initially driven by electrostatic interactions with the PG headgroups and led to the formation of lipid protrusions bulging out from the lipid layer facing the bulk, originating particularly from domain boundaries and membrane defects. The shape, size, and frequency of the lipid protrusions are mainly controlled by the peptide macroscopic properties and the membrane composition. A restructuring of the lipid protrusions into other structures can also occur over time.

Liposomal formulation of retinoids designed for enzyme triggered release.

The design of retinoid phospholipid prodrugs is described based on molecular dynamics simulations and cytotoxicity studies of synthetic retinoid esters. The prodrugs are degradable by secretory phospholipase A(2) IIA and have potential in liposomal drug delivery targeting tumors. We have synthesized four different retinoid phospholipid prodrugs and shown that they form particles in the liposome size region with average diameters of 94-118 nm. Upon subjection to phospholipase A(2), the lipid prodrugs were hydrolyzed, releasing cytotoxic retinoids and lysolipids. The formulated lipid prodrugs displayed IC(50) values in the range of 3-19 microM toward HT-29 and Colo205 colon cancer cells in the presence of phospholipase A(2), while no significant cell death was observed in the absence of the enzyme.

Peptide induced demixing in PG/PE lipid mixtures: a mechanism for the specificity of antimicrobial peptides towards bacterial membranes?

Antimicrobial peptides attract a lot of interest as potential candidates to overcome bacterial resistance. So far, nearly all the proposed scenarios for their mechanism of action are associated with perforating and breaking down bacterial membranes after a binding process. In this study we obtained additional information on peptide induced demixing of bacterial membranes as a possible mechanism of specificity of antimicrobial peptides. We used DSC and FT-IR to study the influence of a linear and cyclic arginine- and tryptophan-rich antimicrobial peptide having the same sequence (RRWWRF) on the thermotropic phase transitions of lipid membranes. The cyclization of the peptide was found to enhance its antimicrobial activity and selectivity ( Dathe, M. Nikolenko, H. Klose, J. Bienert, M. Biochemistry 43 (2004) 9140-9150). A particular preference of the binding of the peptides to DPPG headgroups compared to other headgroups of negatively charged phospholipids, namely DMPA, DPPS and cardiolipin was observed. The main transition temperature of DPPG bilayers was considerably decreased by the bound peptides. The peptides caused a substantial down-shift of the transition of DPPG/DMPC. In contrast, they induced a demixing in DPPG/DPPE bilayers and led to the appearance of two peaks in the DSC curves indicating a DPPG-peptide-enriched domain and a DPPE-enriched domain. These results could be confirmed by FT-IR-spectroscopic measurements. We therefore propose that the observed peptide-induced lipid demixing in PG/PE-membranes could be a further specific effect of the antimicrobial peptides operating only on bacterial membranes, which contain appreciable amounts of PE and PG, and which could in principle also occur in liquid-crystalline membranes.

Hydrophobic interactions are the driving force for the binding of peptide mimotopes and Staphylococcal protein A to recombinant human IgG1.

We studied the interaction of several nona-peptide mimotopes of different sequence and Staphylococcal protein A (SpA) with a recombinant human IgG1 antibody using isothermal titration calorimetry (ITC). The amino acid primary structure of the peptides was varied in order to identify the specific antibody-peptide binding sites. Additionally, the influence of temperature and salt concentration was investigated. An attempt was made to elucidate the structural changes upon complex formation using the determined thermodynamic parameters. The amino acid composition of the mimotopes determined their binding affinity. The binding constant K (a) of the mimotopes was in the range 1 x 10(4) to 1 x 10(6) M(-1). The binding constant of SpA was on the average about three orders of magnitude higher than that of the peptides. The binding constant of the peptides and of SpA decreased with temperature and the binding process was connected with negative changes in enthalpy, entropy, and heat capacity. The binding of the mimotopes to the Fab part of the IgG1 antibody and binding of SpA to the Fc part of the IgG1 antibody were mainly driven by hydrophobic effects and associated with a relatively large change in water-accessible surface area. Determinants for a strong/reduced antibody-peptide binding were identified.