Avidin–biotin-immobilized liposome column for chromatographic fluorescence on-line analysis of solute–membrane interactions

Unilamellar liposomes with entrapped fluorescent dye calcein were stably immobilized in gel beads by avidin–biotin-binding. The immobilized liposomes remained extremely stable upon storage and chromatographic runs. The immobilized calcein-entrapped liposomes were utilized for fluorescent analysis of solute–membrane interactions, which in some cases are too weak to be detected by chromatographic retardation. A liposome column was used as a sensitive probe to detect the interactions of membranes with pharmaceutical drugs, peptides and proteins. Retardation of the solutes was monitored using a UV detector. Perturbation of the membranes, reflected as leakage of the entrapped calcein by some of the solutes, can thus be detected on-line using a flow-fluorescent detector. For the amphiphilic drugs or synthetic peptides, perturbation of membranes became more pronounced when the retardation (hydrophobicity) of the molecules increased. On the other hand, in the case of positively-charged peptides, polylysine, or partially denatured bovine carbonic anhydrase, significant dye leakage from the liposomes was observed although the retardation was hardly to be measured. Weak protein–membrane interactions can thus be assumed from the large leakage of calcein from the liposomes. This provides additional useful information for solute–membrane interactions, as perturbation of the membranes was also indicated by avidin–biotin-immobilized liposome chromatography (ILC).     

Phospholipase A(2)-catalyzed membrane leakage studied by immobilized liposome chromatography with online fluorescent detection

Unilamellar liposomes composed of phosphatidylcholine with an entrapped self-quenching fluorescent dye, calcein, were immobilized in chromatographic gel beads by avidin-biotin binding. Bee venom phospholipase A(2) (PLA(2)) was applied in a small amount onto the immobilized liposome column. The release of calcein from the immobilized liposomes resulting from the catalyzed hydrolysis of the phospholipids was detected online by immobilized liposome chromatography (ILC) using a flow fluorescent detector. The PLA(2)-catalyzed membrane leakage of the immobilized liposomes as studied with ILC was found to be affected by the gel pore size used for immobilization, by liposome size, and as expected by the concentration of calcium, but was unaffected by the flow rate of ILC. The largest PLA(2)-induced calcein release from the liposome column was detected on large unilamellar liposomes immobilized on TSK G6000PW or Sephacryl S-1000 gel in the presence of 1 mM Ca(2+) in the aqueous mobile phase. Comparison with the PLA(2)-catalyzed membrane leakage in free liposome suspensions, we conclude that the fluorescent leakage from liposomes hydrolyzed by PLA(2) can be rapidly and sensitively detected by ILC runs using large amount of immobilized liposomes with entrapped fluorescent dye.     

The elution profile of immobilized liposome chromatography: determination of association and dissociation rate constants

The interaction of lipophilic cations, tetraphenylphosphonium and triphenylphosphonium homologues with liposomes was investigated using immobilized liposome chromatography (ILC). Large unilamellar liposomes with a mean diameter of 100 nm were stably immobilized in chromatographic gel beads by avidin-biotin. The distribution coefficient calculated from (Ve-V0)/Vs (Ve, retention volume; V0, the void volume; Vs, the stationary phase volume) was found to be independent of flow rate, injection amount and gel bed volume, which is consistent with chromatograph theory. The relationship between the bandwidth and solvent flow rate did not follow band-broadening theories reported thus far. We hypothesized that the solvent might be forced to produce large eddies, spirals or turbulent flow due to the presence of liposomes fixed in the gel. Therefore, we developed a new theory for ILC elution: The column is composed of a number of thin disks containing liposomes and solution, and within each disk the solution is well mixed. This theory accounts for our results, and we were able to use it to estimate the rate constants of association and dissociation of the phosphonium to/from liposomes.     

Evaluation of temperature and guanidine hydrochloride-induced protein–liposome interactions by using immobilized liposome chromatography

Hydrophobic interactions between nine model proteins and net-neutral lipid bilayer membranes (liposomes) under stress conditions were quantitatively examined by using immobilized liposome chromatography (ILC). Small or large unilamellar liposomes were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and immobilized in a gel matrix by utilizing covalent coupling between amino-containing lipids and activated gel beads or avidin–biotin biospecific binding. Retardation of bovine carbonic anhydrase (CAB) in ILC was pronounced at particular temperatures (50 and 60 °C) where the local hydrophobicity of theses protein molecules becomes sufficiently large. Protein-induced leakage of a hydrophilic dye (calcein) from immobilized liposomes interior was also drastically enhanced at particular temperatures where large retardation was observed. For other proteins examined, similar results were also observed. The specific capacity factor of the proteins characteristic for the ILC and the amount of calcein released from immobilized liposomes were successfully expressed as a function of the product of the local hydrophobicities of proteins and liposomes, regardless of protein species and the type of the stress conditions applied (denaturant and heating). These findings indicate that lipid membranes have an ability to non-specifically recognize local hydrophobicities of proteins to form stress-mediated supramolecular assemblies with proteins, which may have potential applications in bioprocesses such as protein refolding and separation. ILC was thus found to be a very useful method for the quantitative detection of dynamic protein–liposome interactions triggered by stress conditions.     

Lipophilic drug transfer between liposomal and biological membranes: what does it mean for parenteral and oral drug delivery?

This review presents the current knowledge on the interaction of lipophilic, poorly water soluble drugs with liposomal and biological membranes. The center of attention will be on drugs having the potential to dissolve in a lipid membrane without perturbing them too much. The degree of interaction is described as solubility of a drug in phospholipid membranes and the kinetics of transfer of a lipophilic drug between membranes. Finally, the consequences of these two factors on the design of lipid-based carriers for oral, as well as parenteral use, for lipophilic drugs and lead selection of oral lipophilic drugs is described. Since liposomes serve as model-membranes for natural membranes, the assessment of lipid solubility and transfer kinetics of lipophilic drug using liposome formulations may additionally have predictive value for bioavailability and biodistribution and the pharmacokinetics of lipophilic drugs after parenteral as well as oral administration.     

Covalent immobilization of unilamellar liposomes in gel beads for chromatography

For immobilized (proteo)liposome chromatography, unilamellar liposomes were covalently bound within gel beads that had been activated by CNBr, N-hydroxysuccinimide, tresyl, or chloroformate. Liposomes composed of phosphatidylcholine (PC) and 2 mol% of amino-containing lipid (phosphatidylethanolamine-caproylamine) were immobilized in the activated gels at 5-35 micromol lipid/ml gel and yields of 11-70%. The highest immobilized amount was found in chloroformate-activated TSK G6000PW gel, which contains large pore size (>100 nm). Liposomes composed of PC alone could also be attached to the chloroformate-activated gels at 33-42 micromol/ml gel and yields of 58-65%, probably by crosslinking of the phosphate moiety of phospholipid with the active group of the adsorbent. Liposomes prepared by various phospholipids with or without amino-containing lipids can generally be immobilized in the chloroformate-activated gels. The covalently bound liposomes were characterized by their high stability, unilamellarity, permeability of the membranes, and drug-membrane partition properties. A stable membrane phase was constructed for chromatographic experiments to be performed under extreme elution conditions.     

Lipophilic Drug Transfer Between Liposomal and Biological Membranes: What Does It Mean for Parenteral and Oral Drug Delivery?

This review presents the current knowledge on the interaction of lipophilic, poorly water soluble drugs with liposomal and biological membranes. The center of attention will be on drugs having the potential to dissolve in a lipid membrane without perturbing them too much. The degree of interaction is described as solubility of a drug in phospholipid membranes and the kinetics of transfer of a lipophilic drug between membranes. Finally, the consequences of these two factors on the design of lipid-based carriers for oral, as well as parenteral use, for lipophilic drugs and lead selection of oral lipophilic drugs is described. Since liposomes serve as model-membranes for natural membranes, the assessment of lipid solubility and transfer kinetics of lipophilic drug using liposome formulations may additionally have predictive value for bioavailability and biodistribution and the pharmacokinetics of lipophilic drugs after parenteral as well as oral administration.     

Quantitative affinity chromatographic studies of mitochondrial cytochrome c binding to bacterial photosynthetic reaction center, reconstituted in liposome membranes and immobilized by detergent dialysis and avidin--biotin binding

In order to study the affinity binding of c-type cytochromes to the photosynthetic reaction center (RC) by quantitative affinity chromatography (QAC), RC from Rhodobacter sphaeroides was reconstituted into liposomes composed of egg phosphatidylcholine (EPC) and 2 mol% of biotinyl phosphatidylethanolamine simultaneously as the liposomes were formed and immobilized in (strept)avidin-coupled gel beads by rotary detergent dialysis. The immobilized amount was up to 80 nmol of RC and 33 micromol of lipid/g of moist gel in streptavidin-coupled Sephacryl S-1000 gel. By QAC frontal runs, retardation of mitochondrial cyt c on immobilized RC liposome columns was demonstrated. The dissociation constant for the RC-cyt c interaction was determined to be 0.20-0.57 microM. QAC studies also allowed evaluation of the orientation of reconstituted RC in immobilized liposomes by comparison of the total amount of cyt c binding sites with the amount of available binding sites obtained by QAC. It seems that the RC proteoliposomes immobilized in Sephacryl S-1000 gel exposed the cyt c binding sites on the outer surface of the liposomes due to effects of the gel network pore size and the resulting liposomal size.     

Conformationally changed cytochrome c-mediated fusion of enzyme- and substrate-containing liposomes.

The fusion between enzyme-containing liposomes and substrate-containing liposomes was studied, utilizing conformationally altered cytochrome c as fusion mediator under stress conditions. The liposomes were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and liposome aggregation and subsequent liposome fusion were induced by the addition of cytochrome c, which was partially denatured by 0.5 M guanidinium hydrochloride (GuHCl). In the presence of 0.5 M GuHCl, cytochrome c was found to have a significantly large local hydrophobicity which was determined with the aqueous two-phase partitioning method. Under these conditions, cytochrome c could efficiently bind to POPC bilayer membranes as quantitatively evaluated by immobilized liposome chromatography (ILC). The retardation of cytochrome c treated with 0, 0.5, and 1 M GuHCl on ILC could be correlated with the corresponding local hydrophobicity of cytochrome c. The enzymatic reaction triggered by liposome fusion involved the proteolytic enzyme alpha-chymotrypsin and its substrate succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-AAPF-pNA), which were separately trapped in POPC liposomes. Addition of partially denatured cytochrome c (most likely in the molten globule state) to the mixture of enzyme- and substrate-containing liposomes resulted in the release of one of the hydrolysis products, p-nitroaniline, to the outer phase of the fused liposomes, indicating that the enzymatic reaction occurred during the liposome fusion process. Such a coupled fusion-reaction system may have specific advantages over the conventional fusion analysis and may find application as drug delivery system.     

Immobilization of γ-glutamyl transpeptidase,a membrane enzyme,in gel beads via liposome entrapment

Bovine kidney γ-glutamyl transpeptidase, a membrane enzyme, was immobilized in gel beads by application of the method of Wallstén et al. (Biochim. Biophys. Acta, 982, 47–52, 1989). The gel beads were equilibrated with a dispersion of the enzyme, phospholipids, and cholate and subsequently dialyzed against a buffer for reconstitution and immobilization of enzyme-bound liposomes in the pores of the beads. From the standpoints of the immobilized contents of protein and phospholipids and of the reactivity of γ-glutamyl transpeptidase, a dialysis buffer of Tris-HCl (pH 7.5), a phospholipid concentration of 45 mg/ml in the enzyme-phospholipid-cholate dispersion, and the use of Sepharose CL-6B as the support gel were found to be most appropriate for the immobilization of γ-glutamyl transpeptidase, γ-Glutamyl transpeptidase was activated and stabilized by reconstitution in liposomes. In operation with a packed bed reactor, liposome-bound γ-glutamyl transpeptidase immobilized in Sepharose CL-6B exhibited relatively stable and constant activity for 12 h. In addition, it was found that enzyme substrates were able to pass through the pores of the gel beads to interact with the enzyme present on the outer surface of the liposome membrane in the gel beads. These results thus indicated that a novel support made up of liposomes and Sepharose CL-6B would permit efficient immobilization of lipid-requiring and/or membrane enzymes.     

Stimulation of acid sphingomyelinase activity by lysosomal lipids and sphingolipid activator proteins

Acid sphingomyelinase is a water-soluble, lysosomal glycoprotein that catalyzes the degradation of membrane-bound sphingomyelin into phosphorylcholine and ceramide. Sphingomyelin itself is an important component of the extracellular leaflet of various cellular membranes. The aim of the present investigation was to study sphingomyelin hydrolysis as a membrane-bound process. We analyzed the degradation of sphingomyelin by recombinant, highly purified acid sphingomyelinase in a detergent-free, liposomal assay system. In order to mimic the in vivo intralysosomal conditions as closely as possible a number of negatively charged, lysosomally occuring lipids including bis(monoacylglycero)phosphate and phosphatidylinositol were incorporated into substrate-carrying liposomes. Dolichol and its phosphate ester dolicholphosphate were also included in this study. Bis(monoacylglycero)phosphate and phosphatidylinositol were both effective stimulators of sphingomyelin hydrolysis. Dolichol and dolicholphosphate also significantly increased sphingomyelin hydrolysis. The influence of membrane curvature was investigated by incorporating the substrate into small (SUVs) and large unilamellar vesicles (LUVs) with varying mean diameter. Degradation rates were substantially higher in SUVs than in LUVs. Surface plasmon resonance experiments demonstrated that acid sphingomyelinase binds strongly to lipid bilayers. This interaction is significantly enhanced by anionic lipids such as bis(monoacylglycero)phosphate. Under detergent-free conditions only the sphingolipid activator protein SAP-C had a pronounced influence on sphingomyelin degradation in both neutral and negatively charged liposomes, catalyzed by highly purified acid sphingomyelinase, while SAP-A, -B and -D had no noticeable effect on sphingomyelin degradation.     

Efficiency of mitochondrial uncoupling by modified butyltriphenylphosphonium cations and fatty acids correlates with lipophilicity of cations: Protonophoric vs leakage mechanisms

In order to determine the share of protonophoric activity in the uncoupling action of lipophilic cations a number of analogues of butyltriphenylphosphonium with substitutions in phenyl rings (C₄TPP-X) were studied on isolated rat liver mitochondria and model lipid membranes. An increase in the rate of respiration and a decrease in the membrane potential of isolated mitochondria were observed for all the studied cations, the efficiency of these processes was significantly enhanced in the presence of fatty acids and correlated with the octanol-water partition coefficient of the cations. The ability of C₄TPP-X cations to induce proton transport across the lipid membrane of liposomes loaded with a pH-sensitive fluorescent dye increased also with their lipophilicity and depended on the presence of palmitic acid in the liposome membrane. Of all the cations, only butyl[tri(3,5-dimethylphenyl)]phosphonium (C₄TPP-diMe) was able to induce proton transport by the mechanism of formation of a cation-fatty acid ion pair on planar bilayer lipid membranes and liposomes. The rate of oxygen consumption by mitochondria in the presence of C₄TPP-diMe increased to the maximum values corresponding to conventional uncouplers; for all other cations the maximum uncoupling rates were significantly lower. We assume that the studied cations of the C₄TPP-X series, with the exception of C₄TPP-diMe at low concentrations, cause nonspecific leak of ions through lipid model and biological membranes which is significantly enhanced in the presence of fatty acids.     

A sensitive method for staining proteins transferred to nitrocellulose sheets

A simple physical method to determine the monomer concentration of detergents below as well as above the critical micelle concentration based on the bubble-pressure measurement is described. Aggregated surfactant molecules (micelles) and phospholipid vesicles if present in the sample will not disturb the measurements. Three applications of the method relevant to the preparation of liposomes are shown: (i) measurements of critical micelle concentrations, (ii) evaluation of the affinity constant of the interaction of detergents with liposomal membranes, and (iii) monitoring of residual detergent in liposome preparations during dialysis or after gel chromatography of mixed micelle-derived liposomes. It was found that the efficiency of detergents to produce liposomes during their removal depends on their critical micelle concentrations as well as on their affinity to liposomal membranes.     

Assessing the effects of surfactants on the physical properties of liposome membranes

Knowledge of the partition process of environmentally significant molecules between biological membranes and their surroundings is of vital importance to explain their activity and toxicity, as well as phenomena like absorption, distribution and metabolism. In this research effort, we have studied membrane interactions of three surfactants: t-octylphenoxypolyethoxyethanol (Triton X-100), cetyltrimethylammonium chloride (CTAC) and dodecylbenzene sulphonate (SDBS). Unilamellar liposomes (LUVs) of egg yolk phosphatidylcholine (EPC) were used as membrane models. The partition coefficient, a fundamental parameter in assessing the behaviour of xenobiotic compounds, was determined for SDBS and Triton X-100 by derivative spectrophotometry and fluorescence quenching. The effect of these surfactants upon the physico-chemical characteristics (fluidity, diameter and surface charge) of the liposome membrane was also determined. Results show that all the three surfactants cause an increase in fluidity of the liposome membrane, although for low surfactant concentrations uncharacteristic membrane rigidity was observed, probably due to a change in lipid packing density.     

Interactions of antisense DNA oligonucleotide analogs with phospholipid membranes (liposomes).

Antisense oligonucleotides have the ability to inhibit individual gene expression in the potential treatment of cancer and viral diseases. However, the mechanism by which many oligonucleotide analogs enter cells to exert the desired effects is unknown. In this study, we have used phospholipid model membranes (liposomes) to examine further the mechanisms by which oligonucleotide analogs cross biological membranes. Permeation characteristics of 32P or fluorescent labelled methylphosphonate (MP-oligo), phosphorothioate (S-oligo), alternating methylphosphonate-phosphodiester (Alt-MP) and unmodified phosphodiester (D-oligo) oligodeoxynucleotides were studied using liposomal membranes. Efflux rates (t1/2 values) at 37 degrees C for oligonucleotides entrapped within liposomes ranged from 7-10 days for D-, S- and Alt-MP-oligos to about 4 days for MP-oligos. This suggests that cellular uptake of oligonucleotides by passive diffusion may be an unlikely mechanism, even for the more hydrophobic MP-oligos, as biological effects are observed over much shorter time periods. We also present data that suggest oligonucleotides are unlikely to traverse phospholipid bilayers by membrane destabilization. We show further that MP-oligos exhibit saturable binding (adsorption) to liposomal membranes with a dissociation constant (Kd) of around 20nM. Binding appears to be a simple interaction in which one molecule of oligonucleotide attaches to a single lipid site. In addition, we present water-octanol partition coefficient data which shows that uncharged 12-15 mer MP-oligos are 20-40 times more soluble in water than octanol; the low organic solubility is consistent with the slow permeation of MP-oligos across liposome membranes. These results are thought to have important implications for both the cellular transport and liposomal delivery of modified oligonucleotides.     

Spontaneous transfer of ganglioside GM1 from its micelles to lipid vesicles of differing size.

The spontaneous incorporation of II3-N-acetylneuraminosylgangliotetraosylceramide (GM1) from its micelles into phospholipid bilayer vesicles has been investigated to determine whether curvature-induced changes in membrane lipid packing influence ganglioside uptake. Use of conventional liquid chromatography in conjunction with technically-improved molecular sieve gels permits ganglioside micelles to be separated from phospholipid vesicles of different average size including vesicles with diameters smaller than 40 nm and, thus, allows detailed study of native ganglioside GM1 incorporation into model membranes under conditions where complicating processes like fusion are readily detected if present. At 45 degrees C, the spontaneous transfer rate of GM1 from its micelles to small unilamellar vesicles (SUVs) comprised of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) is at least 3-fold faster than that to similar composition large unilamellar vesicles (LUVs) prepared by octyl glucoside dialysis. Careful analysis of ganglioside GM1 distribution among vesicle populations of differing average size reveals that GM1 preferentially incorporates into the smaller vesicles of certain populations. This behavior is observed in SUVs as well as in LUV-SUV mixtures and actually serves as a sensitive indicator for the presence of trace quantities of SUVs in various LUV preparations. Analysis of the results shows that both differences in the diffusional collision frequency between GM1 monomers and either SUVs or LUVs and curvature-induced changes in the interfacial lipid packing in either SUVs or LUVs can dramatically influence spontaneous ganglioside uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of metal affinity-immobilized liposome chromatography and its basic characteristics

Metal affinity-immobilized liposome chromatography (MA-ILC) was newly developed as a chromatographic technique to separate and analyze peptides. The MA-ILC matrix gel was first prepared by immobilizing liposomes modified with functional ligands. The functional ligand used to adsorb metal ions was N-hexadecyl iminodiacetic acid (HIDA), which is obtained by attaching a long alkyl chain to an iminodiacetic acid (IDA). Cu(II) ion was first adsorbed on the gel matrix through its complex formation with the HIDA on the surface of the immobilized liposome. Synthetic peptides of various types ranging in size from 5 to 40 residues were then used, and their retention properties on the MA-ILC were evaluated. The retention property of peptides on the MA-ILC by using a usual imidazole elution was compared with the retention property in the case of the immobilized metal affinity chromatography (IMAC) and an immobilized liposome chromatography (ILC). It was found that the retention property of peptides on the MA-ILC has the features of both the IMAC and the ILC; the retention ability of peptides depends on both the number of histidine residues in peptides and the liposome membrane affinity of the peptides. Histidine and tryptophan residues among amino acid residues in peptides indicated a high contribution coefficient for the peptide retention on the MA-ILC, probably due to their metal ion and membrane interaction properties, respectively.     

The effect of the placement and total charge of the basic amino acid clusters on antibacterial organism selectivity and potency

Extensive circular dichroism, isothermal titration calorimetry and induced calcein leakage studies were conducted on a series of antimicrobial peptides (AMPs), with a varying number of Lys residues located at either the C-terminus or the N-terminus to gain insight into their effect on the mechanisms of binding with zwitterionic and anionic membrane model systems. Different CD spectra were observed for these AMPs in the presence of zwitterionic DPC and anionic SDS micelles indicating that they adopt different conformations on binding to the surfaces of zwitterionic and anionic membrane models. Different CD spectra were observed for these AMPs in the presence of zwitterionic POPC and anionic mixed 4:1 POPC/POPG LUVs and SUVs, indicating that they adopt very different conformations on interaction with these two types of LUVs and SUVs. In addition, ITC and calcein leakage data indicated that all the AMPs studied interact via very different mechanisms with anionic and zwitterionic LUVs. ITC data suggest these peptides interact primarily with the surface of zwitterionic LUVs while they insert into and form pores in anionic LUVs. CD studies indicated that these compounds adopt different conformations depending on the ratio of POPC to POPG lipids present in the liposome. There are detectable spectroscopic and thermodynamic differences between how each of these AMPs interacts with membranes, that is position and total charge density defines how these AMPs interact with specific membrane models and thus partially explain the resulting diversity of antibacterial activity of these compounds.     

Stabilization of liposomal membranes by thermozeaxanthins: carotenoid-glucoside esters.

Thermozeaxanthins (TZS) are novel carotenoid-glucoside esters existing in the cell membranes of the thermophilic bacterium, Thermus thermophilus. The effect of TZS on membrane permeability was studied by measuring the leakage of the fluorescent dye from calcein-entrapped large unilamellar liposomes (LUVs). The LUVs were composed of a small portion (0.2-1.0 mol%) of TZS and phosphatidylcholine (PC) of various length and saturation degree of hydrocarbon chains. Incorporation of TZS in egg PC LUVs stabilized the liposomes in the temperature range from 30 to 80 degrees C, as only 2.6% of the entrapped calcein leaked out in contrast to 10% release from the egg PC liposomes without TZS. LUVs composed of dipalmitoylphosphatidylcholine (DPPC) or dioleoylphosphatidylcholine (DOPC) were stabilized by the incorporation of TZS at a temperature below 30 degrees C. Inclusion of TZS in LUVs composed of dimyristoylphosphatidylcholine, whose hydrocarbon chains are shorter than both DPPC and DOPC, did not stabilize the liposomes. About 90% of the entrapped dye was lost indicating defects of the liposomal membranes. Matching of the lipid bilayer thickness with the molecular length of TZS in the bilayers is discussed.     

Bilayer interaction and localization of cell penetrating peptides with model membranes: a comparative study of a human calcitonin (hCT)-derived peptide with pVEC and pAntp(43-58)

Cell-penetrating peptides (CPPs) are able to translocate problematic therapeutic cargoes across cellular membranes. The exact mechanisms of translocation are still under investigation. However, evidence for endocytic uptake is increasing. We investigated the interactions of CPPs with phospholipid bilayers as first step of translocation. To this purpose, we employed four independent techniques, comprising (i) liposome buffer equilibrium dialysis, (ii) Trp fluorescence quenching, (iii) fluorescence polarization, and (iv) determination of zeta-potentials. Using unilamellar vesicles (LUVs) of different phospholipid composition, we compared weakly cationic human calcitonin (hCT)-derived peptides with the oligocationic CPPs pVEC and penetratin (pAntp). Apparent partition coefficients of hCT-derived peptides in neutral POPC LUVs were dependent on amino acid composition and secondary structure; partitioning in negatively charged POPC/POPG (80:20) LUVs was increased and mainly governed by electrostatic interactions. For hCT(9-32) and its derivatives, D values raised from about 100-200 in POPC to about 1000 to 1500 when negatively charged lipids were present. Localization profiles of CPPs obtained by Trp fluorescence quenching were dependent on the charge density of LUVs. In POPC/POPG, hCT-derived CPPs were located on the bilayer surface, whereas pVEC and pAntp resided deeper in the membrane. In POPG LUVs, an increase of fluorescence polarization was observed for pVEC and pAntp but not for hCT-derived peptides. Generally, we found strong peptide-phospholipid interactions, especially when negatively charged lipids were present.