Size-induced enhancement of chemical exchange saturation transfer (CEST) contrast in liposomes

Liposome-based chemical exchange saturation transfer (lipoCEST) agents have shown great sensitivity and potential for molecular magnetic resonance imaging (MRI). Here we demonstrate that the size of liposomes can be exploited to enhance the lipoCEST contrast. A concise analytical model is developed to describe the contrast dependence on size for an ensemble of liposomes. The model attributes the increased lipoCEST contrast in smaller liposomes to their larger surface-to-volume ratio, causing an increased membrane water exchange rate. Experimentally measured rates correlate with size, in agreement with the model. The water permeability of liposomal membrane is found to be 1.11 +/- 0.14 microm/s for the specific lipid composition at 22 degrees C. Availability of the model allows rational design of the size of liposomes and quantification of their properties. These new theoretical and experimental tools are expected to benefit applications of liposomes to sensing the cellular environment, targeting and imaging biological processes, and optimizing drug delivery properties.     

Influence of lipid composition on the thermotropic behavior and size distribution of mixed cationic liposomes

Cationic liposomes are studied mainly as nonviral nucleic acid delivery systems and to a lesser extent as carriers/adjuvants of vaccines and as low-molecular-weight drug carriers. It is well established that the performance and the biological activity of liposomes in general are strongly related to their physicochemical properties. We investigated the thermotropic behavior and the size distribution of mixed cationic liposomes formulated with different percentages of 1,2 dimyristoyl-sn-glycero-3-phosphatidylcholine and one of four cationic amphiphiles characterized by a pyrrolidinium headgroup with the aim of achieving a better understanding of how the molecular structure of the cationic amphiphile and its mole percentage affect the physicochemical properties of the liposomes. Multilamellar vesicles and large unilamellar vesicles were studied by differential scanning calorimetry and turbidity, respectively, to characterize the thermotropic behavior and lipid phase, whereas dynamic light scattering was used to determine size distribution. This study shows that subtle modifications in the cationic amphiphile's molecular structure and in liposome composition may have dramatic effects on the organization of the liposome bilayer and hence on the morphological and physicochemical features of the liposomes, thus being highly relevant to the biological features investigated previously.     

Electrically induced deformation of giant liposomes monitored by thickness shear mode resonators

Thickness shear mode resonators are capable of registering small changes in the thickness and viscoelastic properties of ultrathin films attached to their surface. It was found that it is possible to monitor the deformation of surface-bound giant liposomes by applying an electric field with small amplitudes. Changes in the apparent height of attached vesicles in the nanometer range were easily detected as a function of lipid composition. Increasing the bending modulus by adding cholesterol results in a significantly reduced deformation from 16.8 nm (5% cholesterol) down to 3.2 nm (20% cholesterol), rendering this new method a robust and sensitive tool to detect the bending elasticity of liposomes on small length scales. Deformation could be further suppressed by adding anchor groups (biotinylated lipids), resulting in a strongly flattened liposome on an avidin-coated resonator.     

The surface properties of phospholipid liposome systems and their characterisation

The field of liposome (vesicle) research has expanded considerably over the last 30 years. In physical chemical terms liposomes have many of the characteristics of colloidal particles and their stability is determined in part by the classical surface forces. It is now possible to engineer a wide range of liposomes varying in size, phospholipid composition and surface characteristics. The surfaces of liposomes can be modified by the choice of bilayer lipid as well as by the incorporation and covalent linkage of proteins (e.g. antibodies and sugar binding proteins [lectins]), glycoproteins and synthetic polymers. Much of the impetus for liposome design has come from their potential value as drug delivery systems. The development of technologies for the production of such a range of liposome systems has presented interesting problems in the characterisation of their properties. The review addresses the progress that has been made in characterising the surfaces of different types of liposomes with specific reference to their electrophoretic properties and their interpretation and the physical interactions between liposomal bilayers.     

Investigation of Factors Affecting Controlled Release from Photosensitive DMPC and DSPC Liposomes

An investigation of liposomes comprised of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) lipids with cholesterol and zinc phthalocyanine (ZnPC) revealed that several fundamental liposome properties are influenced by composition and by lipid-specific features. DMPC and DSPC liposomes were synthesized, and their compositional changes, encapsulation capacities, morphologies, and release properties were evaluated. In this research, liposome degradation, lysis, and content release were initiated by photolysis, i.e., rupture induced by exposure to light. A controlled release mechanism was created through the introduction of photosensitizers (i.e., ZnPC) embedded within the cholesterol-stabilized liposome membrane. The light wavelength and light exposure time accelerated photodegradation properties of DMPC liposomes compared to DSPC liposomes, which exhibited a slower release rate. Morphological changes in the liposomes were strongly influenced by light wavelength and light exposure time. For both the DMPC and DSPC liposomes, visible light with wavelengths in the red end of the spectrum and broad spectrum ambient lighting (400?C700?nm) were more effective for lysis than UV-A light (365?nm). Heating liposomes to 100?°C decreased the stability of liposomes compared to liposomes kept at room temperatures. In addition, the optimal lipid-to-cholesterol-to-photoactivator ratio that produced the most stable liposomes was determined.     

Liposomes as carriers in electrokinetic capillary chromatography

Liposomes are small membrane-enclosed vesicles composed of either natural or synthetic lipids. Their size can be adjusted on a wide scale and they can be made with well-defined compositions. While liposomes have been extensively used as model biomembranes they have also gained a considerable degree of attention as carriers for drugs as well as for genetic material. The physical properties of liposomes are critically dependent on their chemical composition. In this study liposomes were applied as pseudostationary phases in electrokinetic capillary chromatography. Various negatively charged liposomes, consisting of mixtures of zwitterionic and anionic lipids, were investigated. Major emphasis was put on clarifying the effects of the total lipid concentration, the lipid molar ratio, the lipid head group, and the buffer on the capillary electrophoretic separation of neutral analytes. In addition, the influence of the physical state of the membrane, ie., gel vs. fluid, on the separation was investigated. Corticosteroids were applied as model analytes.     

Individual electrophoretic mobilities of liposomes and acidic organelles displaying pH gradients across their membranes

This report focuses on measuring the individual electrophoretic mobilities of liposomes with different pH gradients across their membrane using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). The results from the individual analysis of liposomes show that, using surface electrostatic theories and the electrokinetic theory as the first approximation, zeta potential contributes more significantly to the electrophoretic mobility of liposomes than liposomal size. For liposomes with an outer pH 7.4 (pH(o) 7.4) and a net negative outer surface charge, the most negative electrophoretic mobilities occur when the inner pH (pH(i)) is 6.8; at higher or lower pH(i), the electrophoretic mobilities are less negative. The theories mentioned above cannot explain these pH-induced electrophoretic mobility shifts. The capacity theory, predicting an induced electrical charge on the surface of liposomes, can only explain the results at pH(i) > 6.8. In this report, we hypothesize that there is a flip-flop process of phospholipids, which refers to the exchange of phospholipids between the outer and inner layers of the membrane. This flip-flop is caused by the pH gradient and membrane instability and results in the observed electrophoretic mobility changes when pH(i) is <6.8. Furthermore, it is found that the mobilities of acidic organelles are consistent with the predictions of liposome models we used here.     

Effects of deformability, uneven surface charge distributions, and multipole moments on biocolloid electrophoretic migration

Liposomes have been widely used as cellular and bioparticle mimics due to their lipid bilayer structure and relative ease of production and manipulation. Such biocolloids are frequently characterized by capillary electrophoresis (CE), which promises a wealth of information about such properties as surface charge, composition, and rigidity. The applicability of this information is somewhat limited, however, since it is interpreted with colloidal theories that do not account for the unique properties of biocolloids. In this work, the effects of deformability, mobile surface charges, intrinsic polarizability, and uneven surface charge distributions are incorporated into colloidal theories in order to better model the electrophoretic behaviors of liposomes.     

Monitoring of membrane collapse and enzymatic reaction with single giant liposomes embedded in agarose gel

Giant liposomes are often used as models for studies on cell membranes. We embedded giant liposomes in agarose gel to fix them for assays. Giant liposomes of dioleoylphosphatidylcholine were embedded in 1% (w/v) agarose gel with a low melting temperature: While only 20–25% of giant liposomes survived embedment, their size distribution was unaffected. Using a confocal laser scanning microscope, we monitored dynamic changes in individual agarose gel-embedded giant liposomes induced by the addition of a surfactant (Triton X-100). The permeation and collapse could be clearly discriminated from each other. Invaginated buds on liposome membranes could also be captured as intermediate structures. Additionally, an enzymatic (β-glucosidase) reaction encapsulated within the target liposome was triggered by the external addition of a non-fluorescent substrate and successfully monitored. These results suggest that embedment in agarose gel is useful for the simple fixation of giant liposomes for biochemical and biophysical assays.     

Interaction of liposomes with cultured cells: possible involvement of lipid peroxidation in cell growth inhibition

Systematic analyses of the interaction between liposomes and cells were examined. Liposomes were found to affect the growth of mouse NIH 3T3 cells depending upon their size, net charge, and cholesterol content. Among the charged compounds, stearylamine was the most inhibitory and showed complete inhibition of cell growth at 100 microM. The cholesterol-rich and small unilamellar vesicles were more suppressive compared to the cholesterol-poor and multilamellar ones, respectively. The binding assay of liposomes to the cells showed a positive correlation between liposome binding and the extent of growth inhibition. Suppression of liposome uptake by inhibitors of the cytoskeletal system and energy metabolism were suggestive of an endocytotic mechanism for the cellular uptake of liposomes. The growth inhibitory effect seemed secondary to the intracellular uptake of liposomes, and peroxidation of incorporated lipids would lead to cellular damage. Therefore, it is highly recommended that potential growth inhibitory effects associated with the particular composition and other properties of liposomes should be carefully assessed in any human studies, especially for long-term use.     

Factors influencing the distribution pattern of porphyrins in cell membranes

The mechanism of the sensitizer-membrane interactions has been studied by following the distribution properties of selected porphyrins, including haematoporphyrin (HP) and protoporphyrin (PP), into unilamellar liposomes of dipalmitoyl phosphatidylcholine (DPPC). The endomembrane distribution of HP and PP has been checked as a function of the membrane fluidity and composition by fluorescence polarization and quenching techniques. At porphyrin concentrations below 0.5 microM, HP and PP exclusively localize in the inner phospholipid monolayer; at higher concentrations, the outer monolayer also becomes populated. The porphyrin binding sites in liposomes, however, are different for HP and PP: HP preferentially distributes into water-accessible lipid regions, while PP localizes in the most hydrophobic loci of the lipid matrix. A porphyrin redistribution occurs when the fluidity properties of the liposomes are changed by addition of cholesterol or cardiolipin. In DPPC-cholesterol vesicles, all HP molecules dissolve in DPPC-rich regions while all PP molecules partition in cholesterol-rich environments. In DPPC-cardiolipin vesicles both porphyrins preferentially localize in regions accessible to the external medium. The effect of the nature of the carrier on porphyrin distribution in membranes has been studied by following the uptake and photosensitization properties of free and DPPC-incorporated PP and HP with rat liver mitochondria. The porphyrin photosensitizing efficiency has been checked by following the impairment of the respiratory function of mitochondria upon irradiation. Liposome-bound HP is less active than aqueous HP in determining membrane photodamage in mitochondria. On the contrary, aqueous PP is a very poor sensitizer as compared to a DPPC liposome-entrapped drug.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multicomponent cationic lipid-DNA complex formation: role of lipid mixing

Multicomponent cationic lipid-DNA complexes (lipoplexes) were prepared by adding linear DNA to mixed lipid dispersions containing two populations of binary cationic liposomes and characterized by means of small angle X-ray scattering (SAXS). Four kinds of cationic liposomes were used. The first binary lipid mixture was made of the cationic lipid (3'[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) and the neutral helper lipid dioleoylphosphocholine (DOPC) (DC-Chol/DOPC liposomes), the second one of the cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the neutral dioleoylphosphatidylethanolamine (DOPE) (DOTAP/DOPE liposomes), the third one of DC-Chol and DOPE (DC-Chol/DOPE liposomes), and the fourth one of DOTAP and DOPC (DOTAP/DOPC liposomes). Upon DNA-induced fusion of liposomes, large lipid mixing at the molecular level occurs. As a result, highly organized mixed lipoplexes spontaneously form with membrane properties intermediate between those of starting liposomes. By varying the composition of lipid dispersions, different DNA packing density regimes can also be achieved. Furthermore, occurring lipid mixing was found to induce hexagonal to lamellar phase transition in DOTAP/DOPE membranes. Molecular mechanisms underlying experimental findings are discussed.     

Quality-by-Design-Based Development of n-Propyl-Gallate-Loaded Hyaluronic-Acid-Coated Liposomes for Intranasal Administration

The present study aimed to develop n-propyl gallate (PG)-encapsulated liposomes through a novel direct pouring method using the quality-by-design (QbD) approach. A further aim was to coat liposomes with hyaluronic acid (HA) to improve the stability of the formulation in nasal mucosa. The QbD method was used for the determination of critical quality attributes in the formulation of PG-loaded liposomes coated with HA. The optimized formulation was determined by applying the Box–Behnken design to investigate the effect of composition and process variables on particle size, polydispersity index (PDI), and zeta potential. Physiochemical characterization, in vitro release, and permeability tests, as well as accelerated stability studies, were performed with the optimized liposomal formulation. The optimized formulation resulted in 90 ± 3.6% encapsulation efficiency, 167.9 ± 3.5 nm average hydrodynamic diameter, 0.129 ± 0.002 PDI, and −33.9 ± 4.5 zeta potential. Coated liposomes showed significantly improved properties in 24 h in an in vitro release test (>60%), in vitro permeability measurement (420 μg/cm²) within 60 min, and also in accelerated stability studies compared to uncoated liposomes. A hydrogen-peroxide-scavenging assay showed improved stability of PG-containing liposomes. It can be concluded that the optimization of PG-encapsulated liposomes coated with HA has great potential for targeting several brain diseases.     

Characterization of solvation properties of lipid bilayer membranes in liposome electrokinetic chromatography

The nature of solute interactions with biomembrane-like liposomes, made of naturally occurring phospholipids and cholesterol, was characterized using electrokinetic chromatography (EKC). Liposomes were used as a pseudo-stationary phase in EKC that provided sites of interactions for uncharged solutes. The retention factors of uncharged solutes in liposome EKC are directly proportional to their liposome-water partition coefficients. Linear solvation energy relationship (LSER) models were developed to unravel the contributions from various types of interactions for solute partitioning into liposomes. Size and hydrogen bond acceptor strength of solutes are the main factors that determine partitioning into lipid bilayers. This falls within the general behavior of solute partitioning from an aqueous into organic phases such as octanol and micelles. However, there exist subtle differences in the solvation properties of liposomes as compared to those of octanol and various micellar pseudo-phases such as aggregates of sodium dodecyl sulfate (SDS), sodium cholate (SC), and tetradecylammonium bromide (TTAB). Among these phases, the SDS micelles are the least similar to the liposomes, while octanol, SC, and TTAB micelles exhibit closer solvation properties. Subsequently, higher correlations are observed between partitioning into liposomes and the latter three phases than that into SDS.     

Adsorbed liposome deformation studied with quartz crystal microbalance

Deformation of surface-adsorbed liposomes is an important parameter that governs the kinetics of their transformations, but one that is very difficult to measure in the case of nm-size liposomes. We investigate the deformation of dimyristoyl phosphatidyl choline liposomes by quartz crystal microbalance (QCM) as a function of temperature and show that it follows the dependence of this lipid's bending modulus on temperature, as expected from theoretical considerations. To corroborate our approach, we model QCM response from adsorbed liposomes by explicitly considering their shape and mechanical properties.     

Membrane interactions of ternary phospholipid/cholesterol bilayers and encapsulation efficiencies of a RIP II protein

Membrane interactions of liposomes of ternary phospholipid/cholesterol bilayers are investigated. These interactions lead to discoidal deformations and regular aggregations and are strongly enhanced by the presence of mistletoe lectin (ML), a RIP II type protein. The encapsulation of ML into liposomal nanocapsules is studied with a systematic variation of the lipid composition to monitor its effect on the physical properties: entrapment, mean size, morphology, and stability. Extrusion of multilamellar vesicles through filters 80 nm pore size was used for the generation of liposomes. The mean sizes of liposomes ranged between 120 and 200 nm in diameter with narrow size distributions. The increase in flow rate with pressure for three dioleoylphosphatidylcholine (DOPC)/cholesterol (Chol)/dipalmitoylphosphatidylcholine (DPPC) lipid mixtures was linear and allowed to extrapolate to the minimum burst pressure of the liposomal bilayers. From the minimum pressures P(min), the bilayer lysis tensions gamma(l) were determined. The increase in P(min) and gamma(l) with an increasing content of a saturated phosopholipid (DPPC) indicates that DPPC increases the mechanical strength of lipid bilayers. Apparently, DPPC, like cholesterol, leads to a less compressible surface and a more cohesive membrane. After preparation, vesicle solutions were purified by gel permeation chromatography to separate encapsulated ML from free ML in the extravesicular solution. Purified liposomes were then characterized. The content of entrapped and adsorbed ML was measured using ELISA. Repetitive freezing/thawing cycles prior to extrusion significantly increased ML uptake. On the contrary, adsorption was not affected neither by lipid composition, nor concentration and preparation. Differences in experimental encapsulation efficiency only reflect the differences in the mean vesicle sizes of the different samples as is revealed by a comparison to a theoretical estimate. Cryo-transmission electron microscopy (Cryo-TEM) images show that beside spherical, single-walled liposomes, there is a considerable fraction of discoidally deformed vesicles. Based on our results and those found in the literature, we speculate that the flattening of the vesicles is a consequence of lipid phase separation and the formation of condensed complexes and areas of different bending elasticities. This phenomenon eventually leads to agglomeration of deformed liposomal structures, becoming more pronounced with the increase in the relative amount of saturated fatty acids, presumably caused by hydrophobic interaction. For the same lipid mixture aggregation correlated linearly with the ML content. Finally, tested liposomal samples were kept at 4 degrees C to examine their stability. Only slight fluctuations in diameter and the increase in polydispersity after 3 weeks of storage occurred, with no statistically significant evidence of drug leakage during a time period of 12 days, illustrating physical stability of liposomes.     

The influence of chondroitin sulfate on composite multilamellar liposomes containing chitosan

A composite multilamellar liposome containing chitosan attached to the inside and outside of the membrane as well as an opposite charged polyelectrolyte, chondroitin, adsorbed at the surface was developed. Not only the chitosan/chondroitin ratio but also the concentration of them were varied. The structure and superficial properties of the liposomes were studied through a combination of light scattering, zeta potential, and small-angle X-rays scattering techniques. While the chitosan/chondroitin ratio affected the superficial charge distributions, the concentration of polyelectrolytes affected the structural properties of the liposomes, as the rigidity of the phospholipid layers. The superficial charge of the resultant composite liposome was influenced by the type and concentration of the polyelectrolyte. Information about the charge density could be obtained by the treatment of zeta potential data, and it was used to estimate the amount of chondroitin adsorbed to the liposome surface. Applying the modified Caillé theory to the X-rays scattering curves, information about the internal structure of the liposomes was accessed. The ability to control the properties of composite multilamellar liposomes is an important issue when they have to be applied as a biomaterial device component.     

Chemically encapsulated structural elements for probing the mechanical responses of biologically inspired systems

A living cell has a crowded environment with a dense distribution of molecules that requires structured organization for its efficient functioning. One component of this structure, the actin cytoskeleton, is essential for providing mechanical support and facilitating many response activities, including the contraction of muscle cells and chemotaxis. Whereas many investigations have provided insight into the mechanical response from either an in vivo or in vitro perspective, a significant gap exists in determining how the living cell response and the polymer physics response are bridged. The understanding of these systems involves studying their components, including the individual cytoskeletal elements versus the higher-order organism organization in a living cell. Here, we leverage this organization in nature by using a chemistry-based approach to mimic the cytoskeleton in an artificial environment composed of spherically distributed lipid bilayers. This construct bears similarities to the cell membrane. To create a structurally regulated environment, we encapsulate G-actin into giant unilamellar vesicles and then polymerize actin filaments within individual liposomes. We visualize these vesicles with epifluorescence microscopy and confocal microscopy. Atomic force microscopy is then used to probe the mechanical properties of these artificial cells. This polymer cytoskeletal network appears to connect with the lipid bilayer and span the internal space within the liposomes in a manner similar to what is observed in living cells. This work will have implications in a variety of fields, including chemistry, polymer physics, structural biology, and engineering mechanics.     

Nanoformulation and Evaluation of Oral Berberine-Loaded Liposomes

Berberine (BBR) is a poorly water-soluble quaternary isoquinoline alkaloid of plant origin with potential uses in the drug therapy of hypercholesterolemia. To tackle the limitations associated with the oral therapeutic use of BBR (such as a first-pass metabolism and poor absorption), BBR-loaded liposomes were fabricated by ethanol-injection and thin-film hydration methods. The size and size distribution, polydispersity index (PDI), solid-state properties, entrapment efficiency (EE) and in vitro drug release of liposomes were investigated. The BBR-loaded liposomes prepared by ethanol-injection and thin-film hydration methods presented an average liposome size ranging from 50 nm to 244 nm and from 111 nm to 449 nm, respectively. The PDI values for the liposomes were less than 0.3, suggesting a narrow size distribution. The EE of liposomes ranged from 56% to 92%. Poorly water-soluble BBR was found to accumulate in the bi-layered phospholipid membrane of the liposomes prepared by the thin-film hydration method. The BBR-loaded liposomes generated by both nanofabrication methods presented extended drug release behavior in vitro. In conclusion, both ethanol-injection and thin-film hydration nanofabrication methods are feasible for generating BBR-loaded oral liposomes with a uniform size, high EE and modified drug release behavior in vitro.