Molecular aspects of the bilayer stabilization induced by poly(l-lysines) of varying size in cardiolipin liposomes

The interaction between poly(l-lysines) of varying size with cardiolipin was investigated via binding assays, X-ray diffraction, freeze-fracture electron microscopy, and ³¹P- and ¹³C-NMR. Binding of polylysines to the lipid only occurred when three or more lysine residues were present per molecule. The strength of the binding was highly dependent on the polymerization degree, suggesting a cooperative interaction of the lysines within the polymer. Upon binding, a structural reorganization of the lipids takes place, resulting in a closely packed multilamellar system in which the polylysines are sandwiched in between subsequent bilayers. Acyl chain motion is reduced in these liquid-crystalline peptide-lipid complexes. From competition experiments with Ca²⁺ it could be concluded that when the affinity of the polylysine for cardiolipin was much larger than that of Ca²⁺, a lamellar polylysine-lipid complex was formed, irrespective of whether an excess of Ca²⁺ was added prior to or after the polypeptide. When the affinity of the polylysine for cardiolipin was less or of the same order as that of Ca²⁺, the lipid was organized in the hexagonal H_II phase in the presence of Ca²⁺. These results are discussed in the light of the peptide specificity of bilayer (de)stabilization in cardiolipin model membranes.     

Aggregation of cardiolipin liposomes induced by monovalent cations

Monovalent ion induced aggregation of the cardiolipin bilayer liposomes is studied. Derived threshold concentrations (Ck) stimulating fast aggregation testify that the order of effectiveness for monovalent cations to cause this process is: H+ greater than Na+ greater than Li+ greater than K+. The Ck is shown to be nonmonotonously dependent on the temperature discovering a maximum in the range approximately 30-40 degrees C. It is also shown that the liposomes preliminary temperature processing for two hours at approximately 70 degrees C as well as the liposomes incubation for several days at approximately 5 degrees C affect the Ck value. In both cases a considerable Ck increase is accompanied by almost two-fold increase of the lipid oxidation index. The studied process is reversible to both electrolyte concentration dilution and temperature changes. However, unlike the phosphatidylserine (PS) and phosphatidic acid (PA) liposomes the observed changes in the cardiolipin case proceeding considerably slower possibly indicate that the potential must be lower in its depth than that in the case of PS and/or PA.     

Peroxidative permeabilization of liposomes induced by cytochrome c/cardiolipin complex

Interaction of cytochrome c with mitochondrial cardiolipin converting this electron transfer protein into peroxidase is accepted to play an essential role in apoptosis. Cytochrome c/cardiolipin peroxidase activity was found here to cause leakage of carboxyfluorescein, sulforhodamine B and 3-kDa (but not 10-kDa) fluorescent dextran from liposomes. A marked decrease in the amplitude of the autocorrelation function was detected with a fluorescence correlation spectroscopy setup upon incubation of dye-loaded cardiolipin-containing liposomes with cytochrome c and H₂O₂, thereby showing release of fluorescent markers from liposomes. The cytochrome c/H₂O₂-induced liposome leakage was suppressed upon increasing the ionic strength, in contrast to the leakage provoked by Fe/ascorbate, suggesting that the binding of cyt c to negatively-charged membranes was required for the permeabilization process. The cyt c/H₂O₂-induced liposome leakage was abolished by cyanide presumably competing with H₂O₂ for coordination with the central iron atom of the heme in cyt c. The cytochrome c/H₂O₂ permeabilization activity was substantially diminished by antioxidants (trolox, butylhydroxytoluene and quercetin) and was precluded if fully saturated tetramyristoyl-cardiolipin was substituted for bovine heart cardiolipin. These data favor the involvement of oxidized cardiolipin molecules in membrane permeabilization resulting from cytochrome c/cardiolipin peroxidase activity. In agreement with previous observations, high concentrations of cyt c induced liposome leakage in the absence of H₂O₂, however this process was not sensitive to antioxidants and cyanide suggesting direct membrane poration by the protein without the involvement of lipid peroxidation.     

Lipid bilayer stabilization of the Na,K-ATPase reconstituted in DPPC/DPPE liposomes

Different subunit aggregates of the Na,K-ATPase may be formed depending on the method used to solubilize and purify the enzyme. We have studied the thermal unfolding of detergent-solubilized and dipalmitoylphosphatidylcholine/ dipalmitoylphosphatidylethanolamine liposome-reconstituted forms of the Na,K-ATPase by circular dichroism (CD) spectroscopy and p-nitrophenylphosphatase activity. The ellipticity at 222 nm of the solubilized and reconstituted forms showed a sigmoid decrease in the absolute value of the signal of 36 and 31% with T(50%) of 44 and 42 degrees C, respectively. The catalytic activity was reduced in two steps with T(50%) of 32 and 52 degrees C in the detergent-solubilized enzyme and T(50%) of 25 and 53 degrees C in the reconstituted enzyme. The reduction in catalytic activity of the detergent-solubilized enzyme was bi-exponential with t(1/2) of 8.3 and 67.9 min, resulting in the total loss of activity after 120 min. However, under the same conditions, the ATPase activity of the reconstituted enzyme was reduced by approx 35% with a t(1/2) of 145 min. The results suggest that the alpha- and beta-subunits present different thermal stability that may be modulated by the nature of the co-solvent (detergent or lipid) used in the preparations of the Na,K-ATPase. In addition, distinct processes of beta-subunit displacement and alpha-alpha-subunit aggregate formation may also contribute to the changes in both the CD spectra and the enzyme activity. Furthermore, we have demonstrated the protective role of the phospholipid bilayer in the reconstituted enzyme compared with the detergent-solubilized enzyme.     

Salt induced B----A transition of poly(dG).poly(dC) and the stabilization of A form by its methylation.

Raman spectra of poly(dG).poly(dC) have been observed in aqueous solutions at various ionic strengths, [NaCl] = 0.03 to 4 M, and at different temperatures, 10 to 60 degrees C. At 30 degrees C, and at [NaCl] = 0.03 M, it was found to have a B-form (with O4'endo-anti guanosine and C2'endo-anti cytidine), whereas, at [NaCl] = 4 M, an A form (with C3'endo-anti guanosine and C3'endo-anti cytidine). At 30 degrees C and [NaCl] = 1 M, namely at an intermediate state, a fraction of this molecules was considered to have a "heteronomous A" form (with O4'endo-anti guanosine and C3' endo-anti cytidine). At 60 degrees C and [NaCl] = 1 M, it assumes the B form, and at 10 degrees C and [NaCl] = 1 M, the A form. Cytosine-5-methylation was found to cause a marked stabilization of the A form. Even at [NaCl] = 0.1 M (at 30 degrees C), a substantial portion of poly(dG).poly(dm5C) was found to have a heteronomous form, in which the dG atrand is in the B form and the dC an A form; it never assumes a complete B form.     

Selective protein interactions with phosphatidylserine containing liposomes alter the steric stabilization properties of poly(ethylene glycol)

Incorporation of 5 mol% poly(ethylene glycol)-conjugated lipids (PEG-lipids) has been shown to extend the circulation longevity of neutral liposomes due to steric repulsion of PEG at the membrane surface. The effects of PEG-lipids on protein interactions with biologically reactive membranes were examined using phosphatidylserine (PS) containing liposomes as the model. Incorporating 15 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG 2000 into PS liposomes resulted in circulation lifetimes comparable to that obtained with neutral liposomes containing 5 mol% DSPE-PEG 2000. These results suggested that 15 mol% DSPE-PEG 2000 may be effective in protecting PS liposomes from the high affinity, PS-mediated binding of plasma proteins. This was determined by monitoring the effects of PEG-lipids on calcium-mediated blood coagulation protein interactions with PS liposomes. Prothrombin binding and procoagulant activity of PS liposomes could be inhibited >80% when 15 mol% DSPE-PEG 2000 was used. These results are consistent with PS on membrane surfaces forming transient nucleation sites for protein binding that may result in lateral exclusion of PEG-lipids incorporated at <10 mol%. These nucleation sites may be inaccessible when PEG-lipids are present at elevated levels where they adopt a highly compressed brush conformation. This suggests that liposomes with reactive groups and PEG-lipids may be appropriately designed to impart selectivity to protein interactions with membrane surfaces.     

Membrane stabilization of vitamin E; interactions of alpha-tocopherol with phospholipids in bilayer liposomes

13C Spin-lattice relaxation times (T1) of 13C-labeled alpha-tocopherol in three kinds of liposomes varying in their contents of arachidoyl residues have been measured by 13C-NMR spectroscopy. On the basis of T1 values, it is proved that the segmental motion of isoprenoid side chain of alpha-tocopherol tends to increase with an increase in the distance from the chromanol moiety, and that three methyl groups attached on the aromatic ring, have some affinity to unsaturated fatty acid residues rather than those of the isoprenoid side chain. These results are incompatible with the hypothesis of Diplock et al. (1) which 4'a- and 8'a-methyl groups of isoprenoid side chain are fitted in the Z-pockets of arachidoyl chain of polyunsaturated lipids in membrane.     

Prevention of peroxidation of cardiolipin liposomes by quinol-based antioxidants

In mammalian mitochondria, cardiolipin molecules are the primary targets of oxidation by reactive oxygen species. The interaction of oxidized cardiolipin molecules with the constituents of the apoptotic cascade may lead to cell death. In the present study, we compared the effects of quinol-containing synthetic and natural amphiphilic antioxidants on cardiolipin peroxidation in a model system (liposomes of bovine cardiolipin). We found that both natural ubiquinol and synthetic antioxidants, even being introduced in micro- and submicromolar concentrations, fully protected the liposomal cardiolipin from peroxidation. The duration of their action, however, varied; it increased with the presence of either methoxy groups of ubiquinol or additional reduced redox groups (in the cases of rhodamine and berberine derivates). The concentration of ubiquinol in the mitochondrial membrane substantially exceeds the concentrations of antioxidants we used and would seem to fully prevent peroxidation of membrane cardiolipin. In fact, this does not happen: cardiolipin in mitochondria is oxidized, and this process can be blocked by amphiphilic cationic antioxidants (Y. N. Antonenko et al. (2008) Biochemistry (Moscow), 73, 1273–1287). We suppose that a fraction of mitochondrial cardiolipin could not be protected by natural ubiquinol; in vivo, peroxidation most likely threatens those cardiolipin molecules that, being bound within complexes of membrane proteins, are inaccessible to the bulky hydrophobic ubiquinol molecules diffusing in the lipid bilayer of the inner mitochondrial membrane. The ability to protect these occluded cardiolipin molecules from peroxidation may explain the beneficial therapeutic action of cationic antioxidants, which accumulate electrophoretically within mitochondria under the action of membrane potential.     

Storage stabilization of enzyme activity by poly(ethyleneimine)

The effect of mixing penicillin acylase with poly(ethyleneimine) is discussed. The properties of the polymer-enzyme system were evaluated for a wide range of enzyme concentrations (0.3–45.5 mg/cm³) and poly(ethyleneimine) concentrations (0.0001–10% wt). It was shown that addition of poly(ethyleneimine) to crude enzyme preparation caused precipitation of ballast protein and stabilization of the enzyme fraction remaining in the supernatant. The soluble fraction had stable activity for 21 days storage at 37 °C while the native enzyme lost about 80% of its initial activity. Additionally, it was ascertained that the polymer very slightly affected the properties of penicillin acylase in the PEI-enzyme preparations. Finally, possible ways of using the polymer-enzyme preparations in a membrane reactor are suggested.This work was supported by Government Committee of Science: Grant KBN # 3 0321 91 1     

Destabilization and stabilization of poly(A) · poly(U) structure by platinum(II) compounds

A methodology for analyzing the intramolecular structural order of the polynucleotide duplex poly(A) · poly(U) has been developed on the basis of molecular biophysics. The combination of circular dichroism spectroscopy and differential scanning calorimetry was shown to be an optimal approach. It ensures the screening of a wide set of substances and interaction conditions and the choice of compound(s) capable of stabilizing the structure and increasing the biological activity of this duplex. The study is aimed at obtaining a new and highly active antiviral drug.     

Destabilization and stabilization of poly(A).poly(U) structure by platinum(II) compounds

On the basis of molecular biophysics, a methodology for the analysis of intramolecular structural order of the polynucleotide duplex poly(A).poly(U) has been developed. It was shown that the combination of circular dichroism spectroscopy with differential scanning calorimetry is an optimal approach, which ensures the screening of a wide set of substances and interaction conditions and the choice of compound(s) that can stabilize the structure and increase the biological activity of this duplex. The study is aimed at obtaining a new and highly active antiviral remedy.     

Molecular aspects on the specific interaction of cytotoxic plant alkaloid palmatine to poly(A)

The interaction of the protoberberine alkaloid palmatine with single and double stranded structures of poly(A) was studied by various biophysical techniques. Comparative binding studies were also performed with double stranded DNA, t-RNA, poly(C)·poly(G), poly(U) and poly(C). The results of competition dialysis, fluorescence, and absorption spectral studies converge to reveal the molecular aspects of the strong and specific binding of palmatine to single stranded poly(A). The binding affinity of palmatine to natural DNA, t-RNA and double stranded poly(A) was weaker while no binding was apparent with single stranded poly(U), poly(C) and double stranded poly(C)·poly(G). The strong affinity of the alkaloid to single stranded poly(A) in comparison to the double stranded structure was also revealed from circular dichroic and viscometric studies. The effect of [Na⁺] ion concentration on the binding process revealed the significant role of electrostatic forces in the complexation. The presence of bound alkaloid also remarkably affected denaturation–renaturation of stacked helical poly(A). The energetics of the strong binding to poly(A) was studied from thermodynamic estimation from van Hoff’ analysis of the temperature dependent binding constants and ultra sensitive isothermal titration calorimertry, both suggesting the binding to be exothermic and enthalpy driven. This study provides detailed insight into the binding specificity of the natural alkaloid to single stranded poly(A) over several other single and double stranded nucleic acid structures suggesting its potential as a lead compound for RNA based drug targeting.     

Vesicle to micelle transitions of egg phosphatidylcholine liposomes induced by nonionic surfactants, poly(oxyethylene) cetyl ethers.

Vesicle to micelle transitions of sonicated liposomes of egg yolk phosphatidylcholine (EPC) induced by a homologous series of nonionic surfactants, poly(oxyethylene) cetyl ethers [POE(n) cetyl ether], were investigated by using the method of turbidity titrations. The turbidities of the mixed dispersions of sonicated vesicles and surfactant were systematically measured as a function of the surfactant added for a wide range of lipid concentrations (from 0.51 to 6.35 mM EPC). From the titration curves, two threshold points representing onset and complete solubilization of liposomal membranes were determined as a probe for the effect of the length of ethylene oxide (EO) moiety on the phase behavior of ternary system of POE(n) cetyl ethers-EPC-excess water. Patterns of turbidity curves and the surfactant concentrations at two threshold points as well as widths of region between two transitions, where lamellar sheets and mixed micelles may coexist, mainly depended on the length of EO head group. With changing the lengths, solubilization of liposomes and phase diagram showed optimal behavior. That is, in the middle range of EO numbers, it resulted in narrowest coexistence region between onset and complete solubilization. Assuming the equilibrium partitioning model, critical effective molar ratios of surfactant to lipid, Rsat, free surfactant concentrations, Dw, and the partition coefficient of surfactant between bilayer and aqueous phase, K, in surfactant-saturated liposomes were quantitatively determined as a function of EO number. Effective ratios, Rsol, and free surfactant concentration in mixed micelles were also determined. In addition, the effects of CMC and HLB of surfactants on the solubilization of liposome were discussed.     

Preparation of pH-sensitive poly(glycidol) derivatives with varying hydrophobicities: their ability to sensitize stable liposomes to pH

We have previously shown that modification with succinylated poly(glycidol) (SucPG) provides stable egg yolk phosphatidylcholine (EYPC) liposomes with pH-sensitive fusogenic property. Toward production of efficient pH-sensitive liposomes, in this study, we newly prepared three carboxylated poly(glycidol) derivatives with varying hydrophobicities by reacting poly(glycidol) with glutaric anhydride, 3-methylglutaric anhydride, and 1,2-cyclohexanedicarboxylic anhydride, respectively, designated as GluPG, MGluPG, and CHexPG. Correlation between side-chain structures of these polymers and their respective abilities to sensitize stable liposomes to pH was investigated. These polymers are soluble in water at neutral pH but became water-insoluble in weakly acidic conditions. The pH at which the polymer precipitated was higher in the order SucPG < GluPG < MGluPG < CHexPG, which is consistent with the number of carbon atoms of these polymers' side chains. Although CHexPG destabilized EYPC liposomes even at neutral pH, attachment of other polymers provided pH-sensitive properties to the liposomes. The liposomes bearing polymers with higher hydrophobicity exhibited more intense responses, such as content release and membrane fusion, at mildly acidic pH and achieved more efficient cytoplasmic delivery of membrane-impermeable dye molecules. As a result, modification with appropriate hydrophobicity, MGluPG, produced highly potent pH-sensitive liposomes, which might be useful for efficient cytoplasmic delivery of bioactive molecules, such as proteins and genes.     

Proteins induced by the double-stranded interferon inducer poly(I).poly(C)

The possible pathways for realization of antiviral activity of interferon inducer poly (I).poly(C) have been studied. The stimulating effect of interferon inducer on the net protein synthesis in human M19 fibroblasts has been demonstrated. Compositions of the specific proteins induced by poly(I).poly(C) or interferon in human M19 fibroblasts and in monkey cells 4647 have been analyzed by electrophoresis technique. The data obtained suggest the existence of common gene products for interferon and ds-inducer. The ds-inducer requires the synthesis of lesser amounts of proteins for realization of its biological activity as compared with interferon.     

Differential uptake of liposomes varying in size and lipid composition by parenchymal and kupffer cells of mouse liver

Using liposomes differing in size and lipid composition, we have studied the uptake characteristics of the liver parenchymal and Kupffer cells. Desferal labeled with iron-59 was chosen as a radiomarker for the liposomal content, because Desferal in its free form does not cross cellular membranes. At various time intervals after an intravenous injection of liposomes into mice, the liver was perfused with collagenase, and the cells were separated in a Percoll gradient. It was found that large multilamellar liposomes (diameter of about 0.5 μm) were mainly taken up by the Kupffer cells. For these large liposomes, the rate of uptake by Kupffer cells was rapid, with maximum uptake at around 2 hours after liposome injection. Unexpectedly, small unilamellar liposomes (diameter of about 0.08 μm) were less effectively taken up by Kupffer cells, and the rate of uptake was slow, with a maximum uptake at about 10 hours after liposome injection. In contrast, parenchymal cells were more effective in taking up small liposomes and the uptake of large liposomes was negligible. In addition, liposomes made with a galactolipid as part of the lipid constituents appeared to have higher affinity to parenchymal cells than liposomes made without the galactolipid. These findings should be of importance in designing suitable liposomes for drug targeting.     

Conformational changes induced in ionized poly(L-arginine) and poly(L-histidine) by sodium dodecyl sulfate

Circular dichroism spectra have been obtained for cationic poly(L -arginine) and poly(L -histidine) in aqueous solutions containing varying amounts of sodium dodecyl sulfate. The detergent induces a disorder-order transition in both polypeptides. In each case the transition is cooperative and occurs when the ratio of detergent to amino acid residue is near unity. The ordered structure formed by poly(L -arginine) is readily identifiable as an α helix. Poly(L -histidine) appears to form a β structure in which the 211-nm electronic absorption band of the imidazole group exhibits significant rotatory strength.     

A left-handed (Z) conformation of poly(dA-dC).poly(dG-dT) induced by polyamines.

Blocks of potential Z-DNA forming alternating purine-pyrimidine (APP) sequences are widely dispersed in native DNAs. We have studied the effects of naturally occurring polyamines on the conformation of a synthetic APP sequence, poly(dA-dC).poly(dG-dT) by circular dichroism spectroscopy. In the presence of micromolar concentrations of spermidine (125 microM) and spermine (16 microM), this polymer undergoes B to Z transition in low ionic strength (2 mM Na+) buffers. The concentration of polyamines required for B to Z transition increases with Na+ in the buffer and a straight line is obtained on plotting ln[Na+] vs. ln [spermidine 3+]. However, at concentrations of polyamines higher than those necessary to induce B to Z transition, Z-DNA converts to psi-DNA, an ordered, twisted, tight packing arrangement of the double helix. These results suggest a pathway for the transient formation of Z-DNA segments in vivo by interaction of the ubiquitous polyamines with naturally occurring blocks of APP sequences.     

Association of hydrophobically-modified poly(ethylene glycol) with fusogenic liposomes

We present results on using cooperative interactions to shield liposomes by incorporating multiple hydrophobic anchoring sites on polyethylene glycol (PEG) polymers. The hydrophobically-modified PEGs (HMPEGs) are comb-graft polymers with strictly alternating monodisperse PEG blocks (M(w)=6, 12, or 35 kDa) bonded to C18 stearylamide hydrophobes. Cooperativity is varied by changing the degree of oligomerization at a constant ratio of PEG to stearylamide. Fusogenic liposomes prepared from N-C12-DOPE:DOPC 7:3 (mol:mol) were equilibrated with HMPEGs. Affinity for polymer association to liposomes increases with the degree of oligomerization; equilibrium constants (given as surface coverage per equilibrium concentration of free polymer) for 6 kDa PEG increased from 6.1+/-0.8 (mg/m(2))/(mg/ml) for 2.5 loops to 78.1+/-12.2 (mg/m(2))/(mg/ml) for 13 loops. In contrast, the equilibrium constant for distearoylphosphatidylethanolamine-poly(ethylene glycol) (DSPE-PEG5k) was 0.4+/-0.1 (mg/m(2))/(mg/ml).The multi-loop HMPEGs demonstrate higher levels of protection from complement binding than DSPE-PEG5k. Greater protection does not correlate with binding strength alone. The best shielding was by HMPEG6k-DP3 (with three 6 kDa PEG loops), suggesting that PEG chains with adequate surface mobility provide optimal protection from complement opsonization. Complement binding at 30 min and 12 h demonstrates that protection by multi-looped PEGs is constant whereas DSPE-PEG5k initially protects but presumably partitions off of the surface at longer times.