POPC Large Unilamellar Vesicles Research Articles

A novel approach to pH-Responsive targeted cancer Therapy: Inhibition of FaDu cancer cell proliferation with a pH low insertion Peptide-Conjugated DGAT1 inhibitor

Targeting enzymes involved in lipid metabolism is increasingly recognized as a promising anticancer strategy. Efficient inhibition of diacylglycerol O-transferase 1 (DGAT1) can block fatty acid (FA) storage. This, in turn, triggers an increase in free polyunsaturated FA concentration, leading to peroxidation and ferroptosis. In this study, we report the development of a pH-sensitive peptide (pHLIP)-drug conjugate designed to selectively deliver DGAT1 inhibitors to cancer cells nested within the acidic microenvironment of tumors. We utilized two previously established pHLIP sequences for coupling with drugs. The study of DGAT1 conjugates in large unilamellar vesicles (LUVs) of different compositions did not reveal enhanced pH-dependent insertion compared to POPC LUVs. However, using in vitro 3D tumor spheroids, significant antiproliferative effects were observed upon exposure to pHLIP-T863 (DGAT1 inhibitor) conjugates, surpassing the inhibitory activity of T863 alone. In conclusion, our study provides the first evidence that pHLIP-based conjugates with DGAT1 inhibitors have the potential to specifically target the acidic compartment of tumors. Moreover, it sheds light on the limitations of LUV models in capturing the pH-dependency of such conjugates.

Liposome-Coated Hydrogel Spheres: Delivery Vehicles with Tandem Release from Distinct Compartments

We have fabricated unilamellar lipid bilayer VESicle-COated hydrogel spheres (VESCOgels) by carbodiimide-mediated coupling of liposomes bearing surface amines to core-shell hydrogel spheres bearing surface carboxyls. The amine-containing moiety, 3-O (2-aminoethoxyethyloxyethyl)carbamyl cholesterol (AECHO), was incorporated into large unilamellar vesicles (LUVs), diameter ∼100 nm, composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The hydrogel, diameter ∼ 1 μm, consisted of a core of poly(N-isopropyl acrylamide) (pNIPAM) and a shell of p(NIPAM-co-acrylic acid (AA)). Activation of these surface-displayed carboxyls with N-hydroxysuccinimidyl (NHS) esters permitted amine coupling upon addition of AECHO-containing POPC LUVs. Bilayer integrity of the hydrogel-bound LUVs was maintained, and fusion of LUVs did not occur. Fluorescence assays of the release of cobalt-calcein trapped within hydrogel-bound LUVs and of sodium fluorescein trapped within the hydrogel itself showed that each compartment retained its distinct release attributes: fast release from the microgel and slow release from the LUVs. It is envisioned that VESCOgels will be useful, therefore, in applications requiring temporally controlled delivery of distinct drugs.

RBPI 21 Causes (HEMI)Fusion and Leakage of Membranes and Promotes Massive Lipopolysaccharide Aggregation

Bacteria are infectious agents that seem to have difficulties in acquiring resistance against natural occurring antimicrobial peptides (AMPs). These positively charged polypeptides were then developed as an alternative to conventional therapy against bacterial infections. rBPI21 is a 21 kDa peptide based on the N-terminal region of the neutrophil bactericidal/permeability-increasing protein (BPI). It has bactericidal effects on Gram-negative bacteria and higher affinity for lipopolysaccharide (LPS), neutralizing its effect. rBPI21 is in phase III clinical trials against meningitis. This work aimed at elucidating the mechanism of action of rBPI21 at the molecular level. We demonstrate that rBPI21 promotes the aggregation of negatively charged large unilamellar vesicles (LUV) and LPS aggregates, by dynamic light scattering, while for zwitterionic phosphatidylcholine (POPC) LUV the size remains unchanged. The aggregation increases with peptide concentration until promotion of massive aggregation followed by sample flocculation/precipitation [1,2]. Through a Förster resonance energy transfer (FRET) assay, the aggregation is followed by (hemi)fusion of the negatively charged vesicles, culminating at their leakage (also assessed by fluorescence spectroscopy). With the rBPI21-lipid interaction, there is a progressive change in the zeta-potential of the LUV systems and LPS aggregates [2]. LUV systems composed of phosphatidylglycerol (POPG) and POPC:POPG mixtures have higher zeta-potential variations than POPC LUV. For the LPS aggregates, rBPI21 neutralizes the surface charge and, at higher peptide concentrations, overcompensates it [2]. The results demonstrate that rBPI21 mechanism of action at the molecular level involves the interaction with the LPS of the outer membrane of Gram-negative bacteria, followed by internalization and leakage induction through the (hemi)fusion of the bacterial outer and inner membranes, both enriched in phosphatidylglycerol. [1] Domingues, MM et al., Biophys. J. 2009, 96, 987-996. [2] Domingues, MM et al., PLoS ONE 2009, 4, e8385.

Dual functions of the human antimicrobial peptide LL-37—Target membrane perturbation and host cell cargo delivery

The mechanisms behind target vs. host cell recognition of the human antimicrobial peptide LL-37 remain ill-defined. Here, we have investigated the membrane disruption capacity of LL-37 using large unilamellar vesicles (LUVs) composed of varying mixtures of POPC, POPG and cholesterol to mimic target and host membranes respectively. We show that LL-37 is unable to induce leakage of entrapped calcein from zwitterionic POPC LUVs, whereas leakage from LUVs partially composed of POPG is fast and efficient. In accordance with typical antimicrobial peptide behavior, cholesterol diminished LL-37 induced leakage. By using linear dichroism and flow oriented LUVs, we found that LL-37 orients with the axis of its induced α-helix parallel to the membrane surface in POPC:POPG (7:3) LUVs. In the same system, we also observed a time-dependent increase of the parallel α-helix LD signal on timescales corresponding to the leakage kinetics. The increased LD may be connected to a peptide translocation step, giving rise to mass balance across the membrane. This could end the leakage process before it is complete, similar to what we have observed. Confocal microscopy studies of eukaryotic cells show that LL-37 is able to mediate the cell delivery of non-covalently linked fluorescent oligonucleotides, in agreement with earlier studies on delivery of plasmid DNA (Sandgren et al., J. Biol. Chem. 279 (2004) 17951). These observations highlight the potential dual functions of LL-37 as an antimicrobial agent against bacterial target cells and a cell-penetrating peptide that can deliver nucleic acids into the host cells.

Relevance of the N-terminal NLS-like sequence of the prion protein for membrane perturbation effects

We investigated the nuclear localization-like sequence KKRPKP, corresponding to the residues 23–28 in the mouse prion protein (mPrP), for its membrane perturbation activity, by comparing effects of two mPrP-derived peptides, corresponding to residues 1–28 (mPrPp(1–28)) and 23–50 (mPrPp(23–50)), respectively. In erythrocytes, mPrPp(1–28) induced ∼ 60% haemoglobin leakage after 30 min, whereas mPrPp(23–50) had negligible effects. In calcein-entrapping, large unilamellar vesicles (LUVs), similar results were obtained. Cytotoxicity estimated by lactate dehydrogenase leakage from HeLa cells, was found to be ∼ 12% for 50 μM mPrPp(1–28), and ∼ 1% for 50 μM mPrPp(23–50). Circular dichroism spectra showed structure induction of mPrPp(1–28) in the presence of POPC:POPG (4:1) and POPC LUVs, while mPrPp(23–50) remained a random coil. Membrane translocation studies on live HeLa cells showed mPrPp(1–28) co-localizing with dextran, suggesting fluid-phase endocytosis, whereas mPrPp(23–50) hardly translocated at all. We conclude that the KKRPKP-sequence is not sufficient to cause membrane perturbation or translocation but needs a hydrophobic counterpart.

Quenching-enhanced fluorescence titration protocol for accurate determination of free energy of membrane binding

Quenching-enhanced fluorescence titration protocol for accurate determination of free energy of membrane binding

Conformation and self-assembly of a nystatin nitrobenzoxadiazole derivative in lipid membranes

Nystatin is a polyene (tetraene) macrolide antibiotic presenting antifungal activity that acts at the cellular membrane level. In the present study, we report the interaction of this antibiotic labelled at its amine group with 7-nitrobenz-2-oxa-1,3-diazole (NBD-Nys) with sterol-free and ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) large unilamellar vesicles (LUV). The mean tetraene to NBD separating distance determined from fluorescence energy transfer measurements increased from 18 to 25.6 Å upon antibiotic binding to the lipid vesicles, indicating that the monomeric labelled antibiotic adopts a more extended conformation in its lipid-bound state than in aqueous solution. The oligomeric state of membrane-bound NBD-Nys was also studied by resonance energy homotransfer between the NBD fluorophores. The decrease measured in its steady state fluorescence anisotropy upon increasing the surface concentration of the NBD-Nys is shown to be consistent with a random distribution of molecules on the surface of the liposomes. This data contradicts the sharp increase measured for nystatin mean fluorescence lifetime in the presence of 10 mol% ergosterol-containing POPC LUV within the same antibiotic concentration range and which is known to report nystatin oligomerization in the lipid vesicles. Therefore, we conclude that the amine group of nystatin is an essential requisite for the supramolecular organization/pore formation of this antibiotic.

An efficient assay for dolichyl phosphate–mannose: protein O-mannosyltransferase

A novel method for quantifying the reaction product from dolichyl phosphoryl mannose:polypeptide mannosyltransferase (protein mannosyl transferase; PMT), was developed. The assay quantifies the amount of radioactivity incorporated into the acceptor peptide YNPTSV from dolichyl phosphoryl [ 3H]mannose (Dol-P-Man). A novel delivery system, large unilamellar vesicles (LUV), composed of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), is used to keep the poorly soluble donor substrate, Dol-P-Man, in solution. The use of LUV allows generation of truly reproducible data and, as an additional benefit, also results in a more than 10 times increase in transfer efficiency. In contrast to the solvent extraction procedures commonly used in previously described PMT assays, the assay reaction product is separated from the radioactive donor substrate on C 18 cartridges. The use of C 18 cartridges allows generation of reproducible data with a low, consistent background and also produces a significant reduction in the time and labor needed for the product workup. In a reaction mixture consisting of 100 μ g POPC LUV, 9×10 5 cpm (approximately 15 pmol) Dol-P-Man, 100 nmol YNPTSV, and aproximately 4 μ g of crude yeast microsomal extract, time-dependent formation of glycosylated product obeys Michaelis-Menten-type kinetics throughout the course of the reaction—until exhaustion of the donor substrate. The linear initial rates of the reaction allowed calculation of an apparent K m of 1 mM, for the acceptor peptide YNPTSV. Variations in detergent concentration in the assay influence transfer efficiency, possibly through interference with the LUV-based donor substrate delivery system. Hence detergent concentrations should be kept constant.

Effects of hemagglutinin fusion peptide on poly(ethylene glycol)-mediated fusion of phosphatidylcholine vesicles.

The effects of hemagglutinin (HA) fusion peptide (X-31) on poly(ethylene glycol)- (PEG-) mediated vesicle fusion in three different vesicle systems have been compared: dioleoylphosphatidylcholine (DOPC) small unilamellar vesicles (SUV) and large unilamellar vesicles (LUV) and palmitoyloleoylphosphatidylcholine (POPC) large unilamellar perturbed vesicles (pert. LUV). POPC LUVs were asymmetrically perturbed by hydrolyzing 2.5% of the outer leaflet lipid with phospholipase A(2) and removing hydrolysis products with BSA. The mixing of vesicle contents showed that these perturbed vesicles fused in the presence of PEG as did DOPC SUV, but unperturbed LUV did not. Fusion peptide had different effects on the fusion of these different types of vesicles: fusion was not induced in the absence of PEG or in unperturbed DOPC LUV even in the presence of PEG. Fusion was enhanced in DOPC SUV at low peptide surface occupancy but hindered at high surface occupancy. Finally, fusion was hindered in proportion to peptide concentration in perturbed POPC LUV. Contents leakage assays demonstrated that the peptide enhanced leakage in all vesicles. The peptide enhanced lipid transfer between both fusogenic and nonfusogenic vesicles. Peptide binding was detected in terms of enhanced tryptophan fluorescence or through transfer of tryptophan excited-state energy to membrane-bound diphenylhexatriene (DPH). The peptide had a higher affinity for vesicles with packing defects (SUV and perturbed LUV). Quasi-elastic light scattering (QELS) indicated that the peptide caused vesicles to aggregate. We conclude that binding of the fusion peptide to vesicle membranes has a significant effect on membrane properties but does not induce fusion. Indeed, the fusion peptide inhibited fusion of perturbed LUV. It can, however, enhance fusion between highly curved membranes that normally fuse when brought into close contact by PEG.

Lipid and stress dependence of amphotericin B ion selective channels in sterol-free membranes

The idea that amphotericin B (AmB) may not require sterols to form ion selective channels has recently been criticized on the grounds that egg phospholipids commonly used in experiments may contain small amounts of sterol which associate with AmB to form AmB/sterol pore channel structures. It was recently shown in this laboratory that modest osmotic stress can enhance the formation of AmB channels in sterol-free egg phosphatidylcholine (eggPC) membranes. We have tested AmB’s ability to form ion channels/defects in synthetic palmitoyl oleoyl (POPC), dieicosenyl (DEPC) and natural eggPC osmotically stressed large unilamellar vesicles (LUV) using pyranine fluorescence detected ion/H + exchange. These sterol-free POPC LUV exhibit greatly increased sensitivity to cation selective AmB channel formation when osmotically stressed; even more than eggPC. Under these stressed conditions, AmB activity was observed at [AmB]/POPC ratios as low as 3.5×10 −4, corresponding to about 34 AmB molecules/vesicle. DEPC vesicles were almost completely unresponsive, demonstrating a strong bilayer thickness dependence. These results prove conclusively that AmB can form sterol-free channels and do so within therapeutic concentration ranges (>0.5–10×10 −6 M) in a stress-dependent manner. This phenomenon may allow us to use osmotic stress changes in simple model systems to spectroscopically isolate and characterize the thus-far elusive AmB channel forming aggregate. In addition, this stress dependence may be responsible for the potentiation of renal toxicity of AmB in the ascending branch of the loop of Henle which is under greatest osmotic stress.

Effects of specific fatty acid acylation of phospholipase A2 on its interfacial binding and catalysis.

Monomeric phospholipase A2 (PLA2) from the venom of Agkistrodon piscivorus piscivorus (App-D49) was treated with 3-acyloxy-4-nitrobenzoic acids to acylate the epsilon-amino groups of two lysines (Lys-7 and Lys-10) in the amino terminal region. Resulting 7,10-diacylated-App-D49s, with acyl groups ranging from lauroyl to palmitoyl, spontaneously aggregated in solution. By contrast, 7,10-dioctanoyl-App-D49 existed as a monomer under the same condition. Kinetic and interfacial binding properties of diacylated enzymes indicated that they catalyzed the hydrolysis at the interface as a monomer. When compared to nonacylated App-D49, diacylated enzymes showed slightly increased activity or decreased activity toward monodispersed 1,2-dibutyryl-sn-glycero-3-phosphocholine, Triton X-100/1,2-dilauroyl-sn-glycero-3-phosphocholine mixed micelles, and small unilamellar vesicles (SUV) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Toward densely-packed liquid-crystalline phospholipid bilayers, such as large unilamellar vesicles (LUV) of POPC, however, diacylated enzymes exhibited a large increase in activity, which reacted up to 250-fold for 7,10-dilauroyl-App-D49 ((kcat/Km)app = (1.0 +/- 0.02) x 10(6) M-1 s-1). Measurements of the penetration of individual diacylated enzymes into 2-oleoyl-3-palmitoyl-sn-glycero-1-phosphocholine (i.e., D-POPC) monolayers indicated that the acyl groups enhanced the interfacial binding of protein by interacting with hydrocarbon moieties of phospholipids and that these hydrophobic interactions remained effective even when the phospholipid packing density was high. Furthermore, fluorometric measurements of the binding of diacylated enzymes to polymerized vesicles of 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphocholine showed that the hydrophobic interactions increased the enzymatic activity toward LUV by accelerating the migration of enzyme molecules to vesicle surfaces. The analysis of the kinetic course of POPC LUV hydrolysis showed that diacylated enzymes as a catalyst were superior to nonacylated App-D49 in that they were not only more catalytically efficient but also able to catalyze more turnovers without being trapped in product-containing vesicles. In summary, the acylation of App-D49 by 3-acyloxy-4-nitrobenzoic acids provides a simple and convenient way of converting the enzyme into a highly active form toward densely-packed liquid-crystalline phospholipid bilayers, which might have potential industrial and biomedical applications.