ABSTRACT
When aqueous salt solutions contain multivalent ions (like Ca2+ or Mg2+), strong correlation effects may lead to ion-bridging, net attraction, and tight-coupling between like-charged interfaces. To examine the effects of surface charge density, temperature, salt type, and salt concentration on the structures of tightly coupled charged interfaces, we have used mixed lipid membranes, containing either saturated or unsaturated tails in the presence of multivalent ions. We discovered that tightly coupled membrane lamellar phases, dominated by attractive interactions, coexisted with weakly coupled lamellar phases, dominated by repulsive interactions. To control the membrane charge density, we mixed lipids with negatively charged headgroups, DLPS and DOPS, with their zwitterionic analogue having the same tails, DLPC and DOPC, respectively. Using solution X-ray scattering we measured the lamellar repeat distance, D, at different ion concentrations, temperatures, and membrane charge densities. The multivalent ions tightly coupled the mixed lipid bilayers whose charged lipid molar fraction was between 0.1 and 1. The repeat distance of the tightly coupled phase was about 4 nm for the DLPS/DLPC mixtures and about 5 nm for the DOPS/DOPC mixtures. In this phase, the repeat distance slightly increased with increasing temperature and decreased with increasing charge density. When the molar fraction of charged lipid was 0.1 or 0.25, a less tightly coupled phase coexisted with the tightly coupled phase. The weakly coupled lamellar phase had significantly larger D values, although they were consistently shorter than the D values in monovalent salt solutions with similar screening lengths.
Subject(s)
Cations, Divalent/chemistry , Cell Membrane/chemistry , Phospholipids/chemistry , Water/chemistryABSTRACT
Large multivesicular liposomes (LMVV) remotely loaded with bupivacaine (Bupisome) were previously demonstrated to be a stable, long-acting local anesthetic. We demonstrate that this is not the case for small unilamellar vesicles (SUV) of the same lipid composition also remotely loaded with bupivacaine. We show that the trapped volume in LMVV is 21-fold higher and the drug-to-lipid mole ratio is 10-fold higher than in SUV. Cryo-transmission electron micrographs and differential interference contrast microscopy show that there are no bupivacaine crystals inside LMVV and SUV. The thermotropic characterization studied by DSC demonstrates that the drug interacts with the liposome membrane, which, together with the above results on the drug-to-lipid ratio, explains the small in vitro drug release from the SUV and large (but <100%) release from the LMVV after 24 h at 37 °C. The absence of analgesia in mice treated locally with SUV loaded with bupivacaine compared with prolonged analgesia from LMVV correlates well with the in vitro results. The study indicates that in LMVV and SUV, part of the bupivacaine is associated with the liposomal membrane, which is poorly available for analgesia. The membrane fraction is very high in SUV and much smaller in LMVV. The much larger trapped volume of the LMVV explains the higher drug availability and better analgesia of LMVV.
ABSTRACT
Recently, developing drug delivery systems exhibiting controlled drug release at the tumor sites emerged as an attractive option for enhancing anticancer therapeutic efficacy. It seems, however, unlikely that single agent therapies will prove effective enough against the myriad cells present within the malignancy. Therefore, next generation nanotherapeutics must not only find their way to the solid tumor but also must effectively destroy the diverse populations of cells promoting tumor growth. Nanoliposomes offer an important advantage in the delivery of a combination of drugs acting synergistically in cancer treatment. They allow controlling the pharmacokinetics and biodistribution of the drugs by uniform time and spatial co-delivery of the agents. However, successful translation of such complex formulations into the clinic relies on understanding critical physicochemical characteristics. These include: liposomes' membrane phase and dynamics, size distribution, state of encapsulated drug, internal environment of liposome, state of grafted polyethylene glycol at the liposome surface, and in-vivo drug release rate. They determine the pharmacokinetics of the formulation and the bioavailability of the drugs. We encapsulated the combination of vincristine (VCR) and topotecan (TPT) in the same nanoliposome (LipoViTo). Our in-vitro and in-vivo characterization of LipoViTo provides an explanation for the good therapeutic efficacy that was previously reported by us. Moreover, we have described how to study these critical features for a two-drug in one nanoliposome formulation. This characterization is an important step for a rational clinical development and for how to ensure liposome product quality of LipoViTo and other liposomal formulations alike.
Subject(s)
Drug Carriers/chemistry , Nanoparticles/chemistry , Polyethylene Glycols/chemistry , Topotecan , Vincristine , Animals , Delayed-Action Preparations , Drug Combinations , Drug Compounding , Drug Stability , Drug Storage , Lipids/chemistry , Liposomes , Mice , Mice, Nude , Particle Size , Solubility , Structure-Activity Relationship , Surface Properties , Tissue Distribution , Topotecan/administration & dosage , Topotecan/pharmacokinetics , Topotecan/therapeutic use , Vincristine/administration & dosage , Vincristine/pharmacokinetics , Vincristine/therapeutic use , Xenograft Model Antitumor AssaysABSTRACT
Like-charged solid interfaces repel and separate from one another as much as possible. Charged interfaces composed of self-assembled charged-molecules such as lipids or proteins are ubiquitous. The present study shows that although charged lipid-membranes are sufficiently rigid, in order to swell as much as possible, they deviate markedly from the behavior of typical like-charged solids when diluted below a critical concentration (ca. 15 wt %). Unexpectedly, they swell into lamellar structures with spacing that is up to four times shorter than the layers should assume (if filling the entire available space). This process is reversible with respect to changing the lipid concentration. Additionally, the research shows that, although the repulsion between charged interfaces increases with temperature, like-charged membranes, remarkably, condense with increasing temperature. This effect is also shown to be reversible. Our findings hold for a wide range of conditions including varying membrane charge density, bending rigidity, salt concentration, and conditions of typical living systems. We attribute the limited swelling and condensation of the net repulsive interfaces to their self-assembled character. Unlike solids, membranes can rearrange to gain an effective entropic attraction, which increases with temperature and compensates for the work required for condensing the bilayers. Our findings provide new insight into the thermodynamics and self-organization of like-charged interfaces composed of self-assembled molecules such as charged biomaterials and supramolecular assemblies that are widely found in synthetic and natural constructs.
Subject(s)
Entropy , Lipid Bilayers/chemistry , Lipids/chemical synthesis , Lipids/chemistry , Macromolecular Substances/chemical synthesis , Macromolecular Substances/chemistry , Surface PropertiesABSTRACT
We apply a means to probe, stabilize, and control the size of lipid raft-like domains in vitro. In biomembranes the size of lipid rafts is ca. 10-30 nm. In vitro, mixing saturated and unsaturated lipids results in microdomains, which are unstable and coalesce. This inconsistency is puzzling. It has been hypothesized that biological line-active surfactants reduce the line tension between saturated and unsaturated lipids and stabilize small domains in vivo. Using solution X-ray scattering, we studied the structure of binary and ternary lipid mixtures in the presence of calcium ions. Three lipids were used: saturated, unsaturated, and a hybrid (1-saturated-2-unsaturated) lipid that is predominant in the phospholipids of cellular membranes. Only membranes composed of the saturated lipid can adsorb calcium ions, become charged, and therefore considerably swell. The selective calcium affinity was used to show that binary mixtures, containing the saturated lipid, phase separated into large-scale domains. Our data suggests that by introducing the hybrid lipid to a mixture of the saturated and unsaturated lipids, the size of the domains decreased with the concentration of the hybrid lipid, until the three lipids could completely mix. We attribute this behavior to the tendency of the hybrid lipid to act as a line-active cosurfactant that can easily reside at the interface between the saturated and the unsaturated lipids and reduce the line tension between them. These findings are consistent with a recent theory and provide insight into the self-organization of lipid rafts, their stabilization, and size regulation in biomembranes.
Subject(s)
Biomimetics/methods , Calcium/metabolism , Chemistry, Physical , Cholesterol/chemistry , Lipid Bilayers/chemistry , Membrane Microdomains/chemistry , Cholesterol/metabolism , Fatty Acids/chemistry , Fatty Acids, Unsaturated/chemistry , Ions/metabolism , Lipid Bilayers/analysis , Membrane Fluidity , Membrane Microdomains/metabolism , Molecular Probes/analysis , Phospholipids/analysis , Phospholipids/chemistry , Scattering, Small Angle , X-RaysABSTRACT
Interactions between charged and neutral self-assembled phospholipid membranes are well understood and take into account temperature dependence. Yet, the manner in which the structure of the membrane is affected by temperature was hardly studied. Here we study the effect of temperature on the thickness, area per lipid, and volume per lipid of charged membranes. Two types of membranes were studied: membranes composed of charged lipids and dipolar (neutral) membranes that adsorbed divalent cations and became charged. Small-angle X-ray scattering data demonstrate that the thickness of charged membranes decreases with temperature. Wide-angle X-ray scattering data show that the area per headgroup increases with temperature. Intrinsically charged membranes linearly thin with temperature, whereas neutral membranes that adsorb divalent ions and become charged show an exponential decrease of their thickness. The data indicate that, on average, the tails shorten as the temperature rises. We attribute this behavior to higher lipid tail entropy and to the weaker electrostatic screening of the charged headgroups, by their counterions, at elevated temperatures. The latter effect leads to stronger electrostatic repulsion between the charged headgroups that increases the area per headgroup and decreases the bilayer thickness.
Subject(s)
Lipid Bilayers/chemistry , Temperature , Entropy , Scattering, Small Angle , Static Electricity , X-Ray DiffractionABSTRACT
In pure water, zwitterionic lipids form lamellar phases with an equilibrium water gap on the order of 2 to 3 nm as a result of the dominating van der Waals attraction between dipolar bilayers. Monovalent ions can swell those neutral lamellae by a small amount. Divalent ions can adsorb onto dipolar membranes and charge them. Using solution X-ray scattering, we studied how the structure of ions and zwitterionic lipids regulates the charge of dipolar membranes. We found that unlike monovalent ions that weakly interact with all of the examined dipolar membranes, divalent and trivalent ions adsorb onto membranes containing lipids with saturated tails, with an association constant on the order of â¼10 M(-1). One double bond in the lipid tail is sufficient to prevent divalent ion adsorption. We suggest that this behavior is due to the relatively loose packing of lipids with unsaturated tails that increases the area per lipid headgroup, enabling their free rotation. Divalent ion adsorption links two lipids and limits their free rotation. The ion-dipole interaction gained by the adsorption of the ions onto unsaturated membranes is insufficient to compensate for the loss of headgroup free-rotational entropy. The ion-dipole interaction is stronger for cations with a higher valence. Nevertheless, polyamines behave as monovalent ions near dipolar interfaces in the sense that they interact weakly with the membrane surface, whereas in the bulk their behavior is similar to that of multivalent cations. Advanced data analysis and comparison with theory provide insight into the structure and interactions between ion-induced regulated charged interfaces. This study models biologically relevant interactions between cell membranes and various ions and the manner in which the lipid structure governs those interactions. The ability to monitor these interactions creates a tool for probing systems that are more complex and forms the basis for controlling the interactions between dipolar membranes and charged proteins or biopolymers for encapsulation and delivery applications.