In vitro and in vivo stability of polymerized mixed liposomes composed of 2,4‐octadecadienoyl groups of phospholipids

The in vitro stability, under freeze–thawing procedures, and in vivo degradation, in rat spleen, of two types of polymerized liposomes were examined: 1,2‐bis‐[2E, ­4E) ‐ octadecadienoyl] ‐ sn ‐ glycero ‐ 3 ‐ phosphocholine (DODPC) and 1‐acyl‐2‐[(2E, 4E)‐octadecadienoyl]‐sn‐glycero‐3‐phosphocholine (AODPC) were used as polymerizable phospholipids. The lipid composition of the liposomes was prepared as DODPC/Chol/SA (Chol = cholesterol, SA = stearicacid), AODPC/Chol/SA (7/7/2 by molar ratio), AODPC/DPPC/Chol/SA (3.5/3.5/7/2 by molar ratio). The liposomes were extruded through a 0.2 µm polycarbonate‐ filter to obtain the approximate particle size of 0.2 µm, and then irradiated with γ‐rays. Hemoglobin‐encapsulated liposomes were also prepared in the same manner with concentrated hemoglobin (Hb) solution. The DODPC/Chol/SA liposome exhibited no trace of particle size change nor Hb leakage. Although not as excellent as the former, the AODPC‐base liposome showed slightly diameter change (below 7.5%) with a substantial abatement of Hb leakage (<3.5%). Transmission electron microscopy observation of spleens also revealed more efficient degradability with AODPC/DPPC/Chol/SA liposome than with DODPC/Chol/SA liposome. Hb‐encapsulated AODPC/DPPC/Chol/SA liposome, after five freeze–thawing cycles, attained an Hb leakage below 3.5% with a particle size change of 0.7–7.5%, and reduced the spleen retention compared with the DODPC‐base liposome. These results suggest that AODPC/DPPC/Chol/SA liposome can be used as a long‐term preservable blood substitute. Copyright © 2000 John Wiley & Sons, Ltd.     

Investigating ligand-receptor interactions at bilayer surface using electronic absorption spectroscopy and fluorescence resonance energy transfer

We investigate interactions between receptors and ligands at bilayer surface of polydiacetylene (PDA) liposomal nanoparticles using changes in electronic absorption spectroscopy and fluorescence resonance energy transfer (FRET). We study the effect of mode of linkage (covalent versus noncovalent) between the receptor and liposome bilayer. We also examine the effect of size-dependent interactions between liposome and analyte through electronic absorption and FRET responses. Glucose (receptor) molecules were either covalently or noncovalently attached at the bilayer of nanoparticles, and they provided selectivity for molecular interactions between glucose and glycoprotein ligands of E. coli. These interactions induced stress on conjugated PDA chain which resulted in changes (blue to red) in the absorption spectrum of PDA. The changes in electronic absorbance also led to changes in FRET efficiency between conjugated PDA chains (acceptor) and fluorophores (Sulphorhodamine-101) (donor) attached to the bilayer surface. Interestingly, we did not find significant differences in UV-vis and FRET responses for covalently and noncovalently bound glucose to liposomes following their interactions with E. coli. We attributed these results to close proximity of glucose receptor molecules to the liposome bilayer surface such that induced stress were similar in both the cases. We also found that PDA emission from direct excitation mechanism was ～2-10 times larger than that of the FRET-based response. These differences in emission signals were attributed to three major reasons: nonspecific interactions between E. coli and liposomes, size differences between analyte and liposomes, and a much higher PDA concentration with respect to sulforhodamine (SR-101). We have proposed a model to explain our experimental observations. Our fundamental studies reported here will help in enhancing our knowledge regarding interactions involved between soft particles at molecular levels.     

Liposome solubilization induced by complexation with dimeric β-cyclodextrin

Dimeric β-cyclodextrins (β-CD) were prepared from the reaction of native β-CD with epichlorohydrin under basic conditions, and the effects on the diacetylene (DA) and polydiacetylene (PDA) liposomes have been investigated. Vesicular DA was solubilized in the presence of dimeric β-CD with the consequent inhibition of polymerization. The result is attributed to the formation of a complex between dimeric β-CD and DA liposomes, and it is clearly differentiated from that of monomeric β-CD. Furthermore, the ordered supramolecular structure of PDA was perturbed by the dimeric β-CD, which was detected from the visible color change. Finally, the morphological characteristics and size of PDA in the absence and presence of dimeric β-CD were examined using transmission electron microscopy and dynamic light scattering The results show fused structure of size more than 200 nm along with the deformation of the vesicles, and they represent a novel phenomenon of liposome structure induced by complexation with dimeric β-CD. The evaluated physicochemical characteristics can be applied to the development of carbohydrate-based detergents.     

Liposome solubilization induced by surfactant molecules in a microchip

The dynamics of liposome solubilization was monitored by dynamic light scattering and optical microscopy. A newly designed Y-shape microchannel connected to a room was incorporated into a microchip and the reaction processes of the liposome suspension and surfactant solution were observed in the room after mixing the two fluids and stopping the flow. By using this microchip, we succeeded in real-time monitoring of liposome solubilization and the following dynamic processes of solubilization were proposed: 1) Deformed liposomes become spherical. 2) The liposome size increases until the surfactant/liposome ratio in the liposome membrane reaches a threshold value. 3) Mixed micelles of surfactants and phospholipids are released and the liposomes collapse.     

Synthesis of fluorocarbon phospholipids and the formation of their liposomes

Two novel types of fluorocarbon phospholipids were prepared with choline and phosphate as the polar head group respectively. They form liposomes under dispersion either by sonication or injection. The size of the liposome with choline group is in the range of 32–37 nm based on the measurements of electron microscopy and of dynamic light scattering. However, the light scattering result showed that the size of the liposome containing phosphate group is larger and increases with the increasing pH value of the system. The fluorocarbon liposome is a much lucid dispersion. It is similar to the conventional hydrocarbon analogues in the ability to incorporate the water-soluble substrates. There is a transition point at 53 °C in the plot of fluorescene intensity of the fluorecent probe ANS incorporated into the fluorocarbon liposome versus temperature.     

Signal enhancement of a micro-arrayed polydiacetylene (PDA) biosensor using gold nanoparticles

Polydiacetylene (PDA) liposomes possess unique properties that allow liposomes to change color and emit fluorescence in response to stimuli such as temperature, antibody-antigen interaction, pH, mechanical stress, and organic solvent. They have been studied extensively as signal transducers in biosensor applications. Here, we describe an antibody-based biosensor using PDA liposomes for detection of human immunoglobulin E (hIgE). Target hIgE chemically bound to hIgE monoclonal antibodies immobilized on PDA liposomes and the fluorescent signals were slightly increased depending on the target protein concentration. As the primary response, the hIgE could be detected to below 10 ng mL(-1). However, fluorescent signals were dramatically increased depending on the target protein concentration when gold nanoparticle-conjugated polyclonal antibody probes were added on the PDA liposomes after the primary immune reaction. A PDA liposome biosensor could detect the hIgE as low as 0.1 ng mL(-1) and the sensitivity was increased up to one hundred times higher than the primary response. As a result, we confirmed that gold nanoparticle-conjugated polyclonal antibody probes efficiently enhanced the fluorescent signal of the PDA liposome biosensor chip. This strategy can be useful to detect proteins of ultra-low concentration.     

Modulating fluorescence resonance energy transfer in conjugated liposomes

We report here a novel system where the rate of energy transfer is based on changes in the spectral overlap between the emission of the donor and the absorption of the acceptor (J) as well as changes in the quantum yield of the acceptor. We use the fluorophore dansyl as the donor and polydiacetylene (PDA) as the acceptor to demonstrate the modulation of FRET through conformationally induced changes in the PDA absorption spectrum following thermal treatment that converts the PDA backbone of the liposome from the blue form to the red form. Energy transfer was found to be significantly more efficient from dansyl to the red-form PDA. These findings support the basis of a new sensing platform that utilizes J-modulated FRET as an actuating mechanism.     

Polydiacetylene liposome arrays for selective potassium detection

Potassium is an important cation in biology, and quantitative detection of the extracellular potassium level is important. However, selective detection of extracellular physiological potassium is a challenging task due to the presence of sodium in a much higher concentration. In this contribution, we describe the development of practical polydiacetylene (PDA) liposome-based microarrays to selectively detect potassium even in the presence of sodium. We utilize the fact that the G-rich ssDNA can fold into a G-quadruplex via intramolecular hydrogen bonding by wrapping around a potassium ion exclusively. We rationally design the PDA liposome in such a way that the G-rich ssDNA probes are presented densely at the liposome surface and form bulky quadruplexes upon binding with K+. The resulting bulky quadruplexes are sterically hindered and repulse each other and impose mechanical stress on the PDA backbone, resulting in the conformational change of the ene-yne backbone of the PDA. As a result, polydiacetylene liposomes turn into the emissive red phase from the nonfluorescent blue phase.     

氟碳磷脂的合成及其脂质体的生成

合成了两种具有氟碳疏水性链的磷脂, 其极性部分分别为胆碱和磷酸根. 超声分散下, 上述两种磷脂均可形成腈质体, 以电子显微镜观察和动态光散射测定得到胆碱形成的脂质体的尺寸在32-37nm左右. 而对后者的光散射测定得出的尺寸较大并与溶液的pH值有关. 具有胆极性基的氟碳脂质体如同相应的碳氢磷脂一样, 具有包容水溶性受物的能力,但透光性更好. 从荧光探针的荧光强度对温度变化的研究指出, 在53℃左右胆碱型氟碳脂质体发生了相变.     

Preparation of polydiacetylene immobilized optically encoded beads

Polydiacetylene (PDA), which can change the chromic and fluorescence properties by inducing environmental perturbations, is immobilized on planar solid supports for many biological applications. In this work, we immobilize PDA onto optically encoded spherical beads (PDA-SERS beads). The prepared PDA immobilized beads (36 μm) exhibit a blue color without fluorescence. By inducing stress, their color and fluorescence properties are changed to red with fluorescence. The SERS spectra of the PDA-SERS beads can be recognized over the PDA background. Moreover, our PDA immobilization methods are successfully applied to silica-surface SERS-encoded beads (5 μm) and proven to also be useful in fluorescence encoding systems.     

LASER MEDIATED RELEASE OF DYE FROM LIPOSOMES

Liposomes made from phospholipids and containing sulforhodamine dye (1-50 mM) have been irradiated with nanosecond and picosecond laser pulses. Individual liposomes were locally heated by laser absorption of dye dimers during a single laser pulse, and heating was sufficient to release the liposome contents. The extent of dye release produced by a single laser pulse was shown to be quantitatively dependent on several interdependent variables, including dye concentration, liposome size, laser excitation parameters and initial temperature of the dye-liposome system. Fluorescence lifetime data having three components have been obtained and analyzed in terms of three dye environments. Quantitative estimates support a photo-induced thermal mechanism for liposome lysis and release of its contents. These results may be useful for laser induced delivery of therapeutic agents or other applications of lasers in biological systems.     

Preliminary Studies on X-Ray-sensitive Liposome

The synthesis of a new type of X-ray-sensitive compound “di-(1-hydroxylundecyl)diselenide” and its application in the preparation of a new type of liposome with X-ray sensitivity was reported.This new ...     

Dynamic and structural aspects of PEGylated liposomes monitored by NMR

Proton-detected NMR diffusion and (31)P NMR chemical shifts/bandwidths measurements were used to investigate a series of liposomal formulations where size and PEGylation extent need to be controlled for ultrasound mediated drug release. The width of the (31)P line is sensitive to aggregate size and shape and self-diffusion (1)H NMR conveys information about diffusional motion, size, and PEGylation extent. Measurements were performed on the formulations at their original pH, osmolality, and lipid concentration. These contained variable amounts of PEGylated phospholipid (herein referred to as PEG-lipid) and cholesterol. At high levels of PEG-lipid (11.5 and 15 mol%) the self-diffusion (1)H NMR revealed the coexistence of two entities with distinct diffusion coefficients: micelles (1.3 to 3x10(-11) m(2)/s) and liposomes (approximately 5x10(-12) m(2)/s). The (31)P spectra showed a broad liposome signal and two distinct narrow lines that were unaffected by temperature. The narrow lines arise from mixed micelles comprising both PEG-lipids and phospholipids. The echo decay in the diffusion experiments could be described as a sum of exponentials revealing that the exchange of PEG-lipid between liposomes and micellar aggregates is slower than the experimental observation time. For low amounts of PEG-lipid (1 and 4.5 mol%) the (31)P spectra consisted of a broad signal typically obtained for liposomes and the diffusion data were best described by a single exponential decay attributed solely to liposomes. For intermediate amounts of PEG-lipid (8 mol%), micellization started to occur and the diffusion data could no longer be fitted to a single or bi-exponential decay. Instead, the data were best described by a log-normal distribution of diffusion coefficients. The most efficient PEG-lipid incorporation in liposomes (about 8 mol%) was achieved for lower molecular weight PEG (2000 Da vs 5000 Da) and when the PEG-lipid acyl chain length matched the acyl chain length of the liposomal core phospholipid. Simultaneously to the PEGylation extent, self-diffusion (1)H NMR provides information about the size of micelles and liposomes. The size of the micellar aggregates decreased as the PEG-lipid content was increased while the liposome size remained invariant.     

ASYMMETRY IN LIPOSOME

Asymmetry of phospholipid distribution along the vertical axis of the bimoiecular membrane in a liposome prepared with soybean phospholipids, PC and PE, was studied by using on ESR technique. From the difference in the values of the order parameter of doxylstearic acid radicals between the sample solutions into which the radicals were added before and after preparation of the liposome, it was concluded that PE is incorporated only in the inner layer of the liposome in the range of 0-30% of PE in mixed PC/PE systems and PE is incorporated in both layers of the liposome over about 30% of PE in the mixture.     

A study on the characterization and stability of rhenium(III) chloride-incorporated liposomes

Nanoliposomes are important carriers capable of packaging drugs for various delivery applications through passive targeting tumor sites by enhancing permeability and retention effect. Radiolabeled liposomes have potential applications in radiotherapy and diagnostic imaging. However, the physico-chemical instability of liposomes during manufacturing and storage limits their extensive application. Therefore, considerable numbers of studies have been made on the stability of liposomes over the last few years in order to overcome this problem. In this study, we attempted to prepare polymer-coated liposomes using water-soluble chitosan in order to enhance the stability of rhenium(III) chloride-incorporated liposomes. They were characterized by an electrophoretic light-scattering spectrophotometer, Fourier transform infrared spectroscopy (FT-IR), UV–Vis spectrometer, and phase-contrast microscopy. The chitosan-coated liposomes are spherical and the particle size is about 800–850 nm. Incorporation of chitosan into the liposome bilayer decreased rhenium(III) chloride release from the liposome due to an increased rigidity of the liposome membrane structure. Chitosan-coated liposomes showed a higher stability compared with the stability of non-coated liposomes. The release characteristics of rhenium(III) chloride encapsulated in the liposome were taken as a measure of stability of the liposome membrane.     

Effect of freezing rate on physical stability of lyophilized cationic liposomes

Factors affecting the storage stability of lyophilized cationic liposomes were investigated using liposomes prepared with various excipients and by different freezing rates, either quick freezing (freezing by immersion into liquid nitrogen) or slow freezing (cooling to -50 degrees C at a rate of -10 degrees C/h). Increases in the particle size of cationic liposomes observed during freeze-drying were inhibited by the addition of sucrose, trehalose and sucrose-dextran mixtures (1 : 1 and 2 : 1 by weight). The storage instability of the formulations, as indicated by changes in particle size, was affected by their glass transition temperature (T(g)). Addition of high-T(g) excipients resulted in smaller increases in the particle size, indicating improvement of storage stability. The storage stability of cationic liposome formulations was also affected by freezing rate. Formulations prepared by slow freezing exhibited better stability. Longer shear relaxation times were observed for formulations prepared by slow freezing compared with those prepared by quick freezing. This indicates that formulations prepared by slow freezing have a lower matrix mobility, which may result in better storage stability. T(g) or (1)H-NMR relaxation measurements could not detect differences in matrix mobility between formulations prepared by different freezing rates. Shear relaxation measurements seem to be a useful method for evaluating the storage stability of cationic liposome formulations.     

Preparation of metallochelating microbubbles and study on their site-specific interaction with rGFP-HisTag as a model protein

The histidine-metallochelating lipid complex is one of the smallest high affinity binding units used as tools for rapid noncovalent binding of histidine tagged molecules, especially recombinant proteins. The advantage of metallochelating complex over protein-ligand complexes (e.g., streptavidine-biotin, glutathiontransferase-glutathion) consists in its very low immunogenicity, if any. This concept for the construction of surface-modified metallochelating microbubbles was proved with recombinant green fluorescent protein (rGFP) containing 6His-tag. This protein is easy to be detected by various fluorescence techniques as flow cytometry and confocal microscopy. Microbubbles (MB) composed of DPPC with various contents of metallochelating lipid DOGS-NTA-Ni were prepared by intensive shaking of the liposome suspension under the atmosphere of sulfur hexafluoride. For this purpose, the instrument 3M ESPE CapMix was used. Various techniques (static light scattering, flow cytometry, and optical microscopy) were compared and used for the measurements of the size distribution of MB. All three methods demonstrated that the prepared MB were homogeneous in their size, and the mean diameter of the MB in various batches was within the range of 2.1-2.8 μm (the size range of 1-10 μm). The presence of large MB (8-10 μm) was marginal. Counting of MB revealed that the average amount of MB prepared of 10 mg of phospholipid equaled approximately 10(9) MB/mL. Lyophilized MB were prepared with saccharose as a cryoprotectant. These MB were shown to be stable both in vitro (the estimated half-live of the MB in bovine serum at 37 °C was 3-7 min) and in vivo (mouse). The stability of the MB was affected by molar content of DOGS-NTA-Ni. DPPC-based metallochelating MB provided a clear and very contrast image of the ventricular cavity soon after the injection. Site selective and stable binding of rGFP-HisTag (as a model of His-tagged protein) onto the surface of metallochelating MB was demonstrated by confocal microscopy.     

Fabrication of nano-scale liposomes containing doxorubicin using Shirasu porous glass membrane

Nano-scale liposomes were successfully produced using a Shirasu porous glass (SPG) membrane emulsification technique. Primary liposomes prepared by a film-hydration method were treated using SPG membranes with different pore sizes (2.0, 1.0, 0.7, 0.5, and 0.2 μm) for control over the liposome size. The liposome sizes were evaluated using a dynamic light scattering method and their morphologies were observed by optical microscopy and transmission electron microscopy. As the passage number of liposomes through SPG membrane increased, the size and its distribution of the liposomes gradually decreased. A smaller pore size of the SPG membrane and a higher applied pressure resulted in liposomes with a smaller size. After the preparation of nano-scale liposomes containing ammonium sulfate (AS), doxorubicin (DOX) was encapsulated in the liposomes by a remote loading method, where AS served as a precipitant for DOX. The encapsulation efficiency of the DOX was maximized up to 94% when the concentrations of AS and DOX were 250 and 0.045 mM, respectively. We have obtained the release profiles of the liposomes with different sizes. As shown below, liposomes with smaller size exhibited a faster release profile of drug due to the large surface area. These nano-scale liposomes encapsulating an anti-cancer drug can potentially be employed as drug delivery vehicles for intravenous injection.     

Enumeration of Liposomes by Multinuclear NMR and Photon Correlation Spectroscopy

Diamagnetic dipalmitoylphosphatidylcholine (DPPC) liposomes dispersed in glucose solution as well as their paramagnetic analogs encapsulating a paramagnetic contrast agent used in magnetic resonance imaging (Gd-HPDO3A, ProHance ® ) were prepared and characterized. The vesicle diameter was assessed by photon correlation spectroscopy (PCS). 31 P NMR spectroscopy was used to measure the phospholipid content and to confirm the highly unilamellar character of the liposome membrane. For both types of liposome preparation, the internal water volume was evaluated below the phase transition temperature ( T m ) by natural abundance 17 O NMR spectroscopy in the presence of a shift reagent confined to the external compartment. For the paramagnetic vesicles, the internal water content was independently assessed by analysis of the biexponential decay of the proton transverse magnetization below T m . Knowing the unilamellarity of the vesicles ( 31 P NMR measurements), the number concentration of liposomes was assessed from the liposomal internal volume calculated from PCS data and the total internal water content obtained by 17 O NMR spectroscopy or 1 H relaxometry. The results obtained are in good agreement and validate these techniques as non invasive methods for the assessment of the number concentration of liposome in suspension.     

Formation and regulation of fullerene-incorporation in liposomes under the phase transition temperature

The fullerene-exchange reaction from a cyclodextrin cavity to liposomes represents one of the best methods to prepare lipid membrane-incorporated [70]fullerenes (C(70)). The C(70)-exchange reaction occurred completely at temperatures above the phase transition temperature (T(m)) of the liposomes; however, lowering the temperature to below the T(m) led to C(70) aggregation outside the liposomes. This observation has limited the development of more functional LMIC(70) using a variety of liposome compositions. In this paper, this reaction was found to occur efficiently by the addition of small amounts of lipids bearing a π-moiety. The π-moieties act as a gate when hydrophobic C(70) migrates into the hydrophilic liposome surface. Therefore, the π-moieties should exist in the polar head groups of the lipids and the C(70)-exchange reaction can be controlled by pH.