RESUMO
Viscosin is a membrane-permeabilizing, cyclic lipopeptide (CLiP) produced by Pseudomonas species. Here, we have studied four synthetic analogs (L1W, V4W, L5W, and L7W), each with one leucine (Leu; L) or valine residue exchanged for tryptophan (Trp; W) by means of time-resolved fluorescence spectroscopy of Trp. To this end, we recorded the average fluorescence lifetime, rotational correlation time and limiting anisotropy, dipolar relaxation time and limiting extent of relaxation, rate constant of acrylamide quenching, effect of H2O-D2O exchange, and time-resolved half-width of the spectrum in the absence and presence of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes. Structure, localization, and hydration of the peptides were described by molecular dynamics simulations. The combination of the parameters provides a good description of the molecular environments of the Trp positions and the behavior of viscosin as a whole. Of particular value for characterizing the impact of viscosin on the membrane is the dipolar relaxation of Trp4 in V4W, which is deeply embedded in the hydrophobic core. The limiting relaxation level represents the membrane perturbation-unlike typical membrane probes-at the site of the perturbant. Fractions of Trp4 relax at different rates; the one not in contact with water upon excitation relaxes via recruitment of a water molecule on the 10-ns timescale. This rate is sensitive to the concerted membrane perturbation by more than one lipopeptide, which appears at high lipopeptide concentration and is assumed a prerequisite for the final formation of a membrane-permeabilizing defect. Trp7 relaxes primarily with respect to neighboring Ser residues. Trp5 flips between a membrane-inserted and surface-exposed orientation.
RESUMO
Micellar drug delivery systems (MDDS) for the intravenous administration of poorly soluble drugs have great advantages over alternative formulations in terms of the safety of their excipients, storage stability, and straightforward production. A classic example is mixed micelles of glycocholate (GC) and lecithin, both endogenous substances in human blood. What limits the use of MDDS is the complexity of the transitions after injection. In particular, as the MDDS disintegrate partially or completely after injection, the drug has to be transferred safely to endogenous carriers in the blood, such as human serum albumin (HSA). If this transfer is compromised, the drug might precipitateâa process that needs to be excluded under all circumstances. The key question of this paper is whether the high local concentration of GC at the moment and site of MDDS dissolution might transiently saturate HSA binding sites and, hence, endanger quick drug transfer. To address this question, we have used a new approach, which is time-resolved fluorescence spectroscopy of the single tryptophan in HSA, Trp-214, to characterize the competitive binding of GC and the drug substitute anilinonaphthalenesulfonate (ANS) to HSA. Time-resolved fluorescence of Trp-214 showed important advantages over established methods for tackling this problem. ANS has been the standard "model drug" to study albumin binding for decades, given its structural similarity to the class of naphthalene-containing acidic drugs and the fact that it is displaced from HSA by numerous drugs (which presumably bind to the same sites). Our complex global fit uses the critical approximation that the average lifetimes behave similarly to a single lifetime, but the resulting errors are found to be moderate and the results provide a convincing explanation of the, at first glance, counterintuitive behavior. Accordingly, and largely in line with the literature, we observed two types of sites binding ANS at HSA: 3 type A, rather peripheral, and 2 type B, likely more central sites. The latter quench Trp-214 by Förster Resonance Energy Transfer (FRET) with a rate constant of ≈0.4 ns-1 per ANS. Adding millimolar concentrations of GC displaces ANS from the A sites but not from B sites. At incomplete ANS saturation, this causes a GC-induced translocation of ANS from A to the more FRET-active B sites. This leads to the apparent paradox that the partial displacement of ANS from HSA increases its quenching effect on Trp-214. The most important conclusion is that (ANS-like) drugs cannot be displaced from the type-B sites, and consequently, drug transfer to these sites is not impaired by competitive binding of GC in the vicinity of a dissolving micelle. The second conclusion is that for unbound GC above the CMC (9 mM), ANS equilibrates between HSA and GC micelles but with a strong preference for free sites on HSA. That means that even persisting micelles would lose their cargo readily once exposed to HSA. For all MDDS sharing this property, targeted drug delivery approaches involving them as the nanocarrier would be pointless.
Assuntos
Sistemas de Liberação de Medicamentos , Micelas , Albumina Sérica Humana , Tensoativos , Humanos , Sítios de Ligação , Sistemas de Liberação de Medicamentos/métodos , Albumina Sérica Humana/química , Albumina Sérica Humana/metabolismo , Tensoativos/química , Espectrometria de Fluorescência , Naftalenossulfonato de Anilina/química , Ligação ProteicaRESUMO
Lysolipids such as lauroyl, myristoyl, and palmitoyl lysophosphatidylcholine (LPC) insert into the outer leaflet of liposomes but do not flip to the inner leaflet over many hours. This way, they create asymmetry stress between the intrinsic areas of the two leaflets. We have studied how this stress is relaxed with particular emphasis on the budding and fission of small (diameter 20-30 nm) daughter vesicles (DVs). Asymmetric flow field-flow fractionation was utilized to quantify the extent of budding from large unilamellar vesicles after exposure to LPC. Budding starts at a low threshold of the order of 2 mol% LPC in the outer (and ≈0 mol% LPC in the inner) leaflet. We see reason to assume that the fractional fluorescence intensity from DVs is a good approximation for the fraction of membrane lipid, POPC, transferred into DVs. Accordingly, budding starts with a "budding power" of ≈6 POPC molecules budding off per LPC added, corresponding to a more than 10-fold accumulation of LPC in the outer leaflet of DVs to ≈24 mol%. As long as budding is possible, little strain is built up in the membranes, a claim supported by the lack of changes in limiting fluorescence anisotropy, rotational correlation time, and fluorescence lifetime of symmetrically and asymmetrically inserted TMA-DPH. At physiological osmolarity, budding is typically limited to 20-30% of budded fraction with some batch-to-batch variation, but independent of the LPC species. We hypothesize that the budding limit is determined by the excess area of the liposomes upon preparation, which is then used up upon budding given the larger area-to-volume ratio of smaller liposomes. As the mother vesicles approach ideal spheres, budding must stop. This is qualitatively supported by increased and decreased budding limits of osmotically predeflated and preinflated vesicles, respectively.