Liposome Solution Research Articles

Transdermal delivery of a pineal hormone: Melatonin via elastic liposomes

Melatonin (MT) is a good candidate for transdermal delivery considering its short biological half-life, low molecular weight and a variable oral absorption. The objective of this work was to develop a novel formulation of melatonin for its efficient transdermal delivery. Melatonin loaded elastic liposomal formulation was prepared, characterized and the effect of this developed formulation on the in vitro permeation of melatonin across human cadaver skin was investigated, using a locally fabricated Franz diffusion cell. Skin permeation potential of the developed formulation was assessed using confocal laser scanning microscopy (CLSM), which revealed an enhanced permeation of the formulation to the deeper layers of the skin (up to 180 μm) following channel like pathways. Skin permeation profile of melatonin through elastic liposomal formulations was observed and the investigations revealed an enhanced transdermal flux (51.2±2.21 μg/cm 2/h), decreased lag time (1.1 h) and an optimum permeability coefficient (15.06±0.52 cm/h) for melatonin. The obtained flux was nearly 5 and 12.3 times higher than conventional liposomal and plain drug solution, respectively ( P<0.005). Our result suggests the feasibility of elastic liposomal system for transdermal delivery of melatonin thereby eliminating the limitations of long lag time and poor skin permeation associated with the drug.

Chondroitin 6-sulfate and dextran sulfate promote hypochlorite-induced peroxidation of phosphatidylcholine liposomes

In this work, we studied whether chondroitin sulfates and dextran sulfates (DXSs) can influence hypochlorite-induced peroxidation of phosphatidylcholine (PC) liposomes. Multilamellar liposomes (2 mg lipid/ml) were prepared in phosphate buffer, pH 7.4, with NaCl or not and exposed to reagent HOCl/ClO − (1 mM) at 37 °C in the presence of different concentrations of chondroitin 6-sulfate (C6S), chondroitin 4-sulfate (C4S), DXS 8000, DXS 40,000, and DXS 500,000. Lipid peroxidation was assessed by thiobarbituric acid-reactive substance (TBARS) production. DXSs and C6S enhanced TBARS production in a dose-dependent manner. The decline in TBARS production at the relatively high C6S concentrations may be attributed to C4S present in C6S, since in contrast to C6S, C4S is known to react with hypochlorite. Dextrans, nonsulfated analogues of DXS, failed to modulate TBARS production. This fact indicates the important role of negatively charged sulfate groups for DXS to facilitate hypochlorite-induced peroxidation of PC liposomes. The electrostatic nature of the mechanism providing for the pro-oxidative effect of DXS was also supported by the influence of liposome surface charge and solution ionic strength on the extent of liposome peroxidation. The addition of calcium ions to the incubation mixture did not prevent the pro-oxidative action of DXS. The relevance of the results to atherogenesis is discussed.

Immobilization of liposomes onto quartz crystal microbalance to detect interaction between liposomes and proteins

To study the interaction between liposomes and proteins, intact liposomes were immobilized on a metal planar support by chemical binding and/or bioaffinity using a quartz crystal microbalance (QCM). A large decrease in the resonance frequency of quartz crystal was observed when the QCM, modified by a self-assembled monolayer (SAM) of carboxythiol, was added to liposome solutions. The stable chemical immobilization of intact liposomes onto SAM was judged according to the degree with which adsorbed mass depended on the prepared size of liposomes, as well as on the activation time of SAMs when amino-coupling was introduced, where the liposome coverage of electrodes was 69 ± 8 % in optimal conditions. When avidin–biotin binding was used on amino-coupling liposome layers, liposome immobilization finally reached 168% coverage of the electrode surface. Denatured protein was also successfully detected according to the change in the frequency of the liposome-immobilized QCM. The adsorbed mass of denatured carbonic anhydrase from bovine onto immobilized liposomes showed a characteristic peak at a concentration of guanidine hydrochloride that corresponded to a molten globule-like state of the protein, although the mass adsorbed onto deactivated SAM increased monotonously.

Micelles as Intermediates in the Preparation of Protein−Liposome Conjugates

A method for preparing protein-liposome conjugates based on micelles as intermediates was developed. Ovalbumin was thiolated with 2-IT and conjugated to the surface of micelles composed of a maleimide-derivatized active lipid and a micelle-forming lipid. These micelles were then incubated with liposomes, allowing the micelle components to exchange into the liposome bilayers. Using this technique we were able to demonstrate that it was possible to saturate the surface of the micelle with protein and use this property to control the level of conjugation. Titration of these protein-micelle conjugates into liposome solutions resulted in reproducible batches of protein-liposome conjugates. Chemical cross-linking could be observed in some cases; however, this was controllable through selection of reagent concentrations. The effects of parameters such as thiolation levels, micelle lipid composition, active lipid structure, micelle-forming lipid structure, and micelle/liposome/protein ratios were examined. The method represents a general approach to the preparation of well defined and reproducible protein-liposome-based drug formulations.

Polydiacetylene Liposomes Attached to Glass Fibers for Fluorescent Bioassays

ABSTRACTPolydiacetylene (PDA) is a conjugated polymer that changes its fluorescent state in response to environmental changes and can act as a transducer to convert molecular interactions into a discernable output measurable in the macroscopic world. Energy transfer to fluorophores can further enhance the fluorescent signal. Diacetylene liposomes can be prepared with phospholipids and other cell membrane components in the liposomes, and photopolymerized. PDA liposomes have been used for absorbance based detection of biological targets; however, moving to fluorescence detection gives increased sensitivity and also allows sensing from PDA structures deposited on opaque membranes. Unfortunately, PDA liposomes are prone to aggregation, particularly in the presence of divalent cations. Many enzymes require divalent cations such as Mg2+, Mn2+, Ca2+, etc., as co-factors; the tendency of PDA liposomes to aggregate in the presence of these cations limits their use as a platform for detection of enzymatic activity. We have developed methods for attaching PDA liposomes to glass fiber membranes, via thiol-epoxide coupling chemistry, for use in bio-assays and have seen that these materials can be used in place of PDA liposome solutions. We present here the attachment of PDA liposomes to glass fiber membranes in 96-well format, cryogenic TEM scans of the attached liposomes and the use of these materials in fluorescence assays to detect the activity and inhibition of phospholipase A2.

Liposomal Formulations of Inflammatory Bowel Disease Drugs: Local versus Systemic Drug Delivery in a Rat Model

Based on adherence to intestinal mucosa, intralumenally administered liposomal formulations of 5-aminosalicylate (5-ASA) and 6-mercaptopurine (6-MP) were studied for their potential to enhance local drug delivery to intestinal tissue for the treatment of inflammatory bowel disease. 5-ASA was encapsulated in standard phospholipid liposomes while 6-MP required encapsulation in nonphospholipid liposomes to obtain equivalent drug loading. Encapsulation efficiency was measured by size-exclusion chromatography/high-performance liquid chromatogtaphy (HPLC). Liposomal formulations or solution of the drugs were injected into unligated jejunum to compare pharmacokinetics and into ligated loops of rat ileum and colon to evaluate local delivery. Dextran sulfate and acetic acid induced colitis were used as models of lower intestinal inflammation. Plasma, tissue and luminal drug and metabolite levels were measured by liquid scintillation counting or HPLC. Encapsulation efficiency of 6-MP was dependent on lipid content and composition. While liposomal encapsulation significantly reduced systemic absorption of 5-ASA this was not the case for 6-MP. Liposomal adherence to intestinal tissue resulted in increased tissue levels for 5-ASA; however, 6-MP local tissue levels were not improved compared to solution drug. Nonphospholipid liposomes optimize encapsulation of 6-MP. While liposomal formulations show potential for local drug delivery to diseased bowel, drug physicochemical properties, absorption, and metabolic profiles dictate tissue-targeting potential. Liposomes reduce systemic availability from paracellular absorption of hydrophilic 5-ASA, but fail to improve local tissue delivery of 6-MP, a molecule absorbed by passive membrane permeation that undergoes extensive first- pass metabolism.

Giant liposomes in physiological buffer using electroformation in a flow chamber

We describe a method to obtain giant liposomes (diameter 10–100 μm) in solutions of high ionic strength to perform a membrane-binding assay under physiological conditions. Using electroformation on ITO electrodes, we formed surface-attached giant liposomes in solutions of glycerol in a flow chamber and then introduced solutions of high ionic strength (up to 2 M KCl) into this chamber. The ionic solution exchanged with the isoosmolar glycerol solution inside and outside the liposomes. An initial mismatch in index of refraction between the inside and outside of liposomes allowed for the observation of solution replacement. Ions and small polar molecules exchanged into and out of surface-attached liposomes within minutes. In contrast, liposomes formed in solutions of macromolecules retained molecules larger than 4 kDa, allowing for encapsulation of these molecules for hours or days even if the solution outside the liposomes was exchanged. We propose that solutes entered liposomes through lipid tubules that attach liposomes to the film of lipids on the surface of the ITO electrode. The method presented here makes it straightforward to perform flow-through binding assays on giant liposomes under conditions of physiological ionic strength. We performed a membrane-binding assay for annexin V, a calcium-dependent protein that binds to phosphatidylserine (PS). The binding of annexin V depended on the concentration of PS and decreased as ionic strength increased to physiological levels.

동결방지제 첨가에 의한 리포솜 나노분말의 안정성

The addition of cryoprotectants as liposome stabilizes has been used to increase the stability of the redispersed liposomes in solution. We have studied the protective effect of cryoprotectants such as saccharides and amino acid derivatives on the storage stability of the liposomes when the liposome powders were redispersed in solution after freezedrying. The liposome size before and after freeze-drying was measured to evaluate the liposome stability. Experimental conditions to maintain the stability of liposomes was optimized in procedure of redipersion with the cryoprotectants. As a result, the size of liposome was maintained in 100±5 nm with the addition of maltose or sucrose at 37 C for 30 days, which shows the enhanced stability during the freeze-drying.

Piperazine-based buffers for liposome coating of capillaries for electrophoresis

Anionic liposomes can be coated on fused-silica capillaries for electrophoresis in the presence of N-(hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) as background electrolyte (BGE) solution. In this work, the interaction of various compounds with zwitterionic and anionic phospholipid coatings was studied with HEPES at pH 7.4 as BGE solution. The chromatographic and electrophoretic behavior of three test sample solutions (anionic, cationic, and neutral) was investigated for evaluation of the phospholipid coatings. Our results show that hydrophobic interactions between analytes and the phospholipid coating are important for the migration of charged analytes. In addition, the performances of other piperazine-based buffers, i.e., N-(2-hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid), piperazine-N,N'-bis(2-ethanesulfonic acid), and piperazine-N,N'-bis(hydroxypropane sulfonic acid), at pH 7.4, as liposome solvent and BGE solution were evaluated and compared with the performance of HEPES at pH 7.4. The anionic liposome solution comprised 80/20 mol% phosphatidylcholine/phosphatidylserine. A simple test solution was selected and the chromatographic and electrophoretic migration behavior of the analytes was evaluated. The results show that, in addition to HEPES, other piperazine-based buffers at pH 7.4 are suitable for coating of fused-silica capillaries with anionic liposomes.

Emulsified Orange Oil in an Aqueous Vesicle Solution

Dilute emulsions of orange oil‐in‐water were prepared by sonication of the oil into water and into a prepared liposome solution. Emulsion droplet size and vapor pressure of the main oil component were determined. The results showed some intriguing features of particle size and vapor pressure, which are rationalized based on the association structures in flavor systems with surfactants and water.

Identification of Lipid Aggregate Structures on TiO2 Surface Using Headgroup IR Bands

Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy was used to study the nature of the dipalmitoylphosphatidylcholine (DPPC) aggregated structures adsorbed on TiO(2). DPPC molecules were assembled on TiO(2) using Langmuir-Blodgett (LB) deposition methods or by directly flowing the DPPC liposome solution across the TiO(2)-coated ATR crystal. We found that there is a direct correlation between the intensity and frequency position of the zwitterionic headgroup IR bands and the nature of LB films. Specifically, we have shown that the bands due to PO(2)(-) modes are sensitive to changes in the degree of hydration of the LB films and that the symmetric deformation vibrational mode (delta(s) (+)N-CH(3)) is sensitive to interaction with oppositely charged surface sites. Using this information, we found that the liposomes adsorbed on TiO(2) remain intact as vesicles and that the vesicles are stable and not removed in flowing water. We have also shown that the antisymmetric deformation vibrational (delta(as) (+)N-CH(3)) modes are sensitive to changes in lateral-lateral DPPC interactions. This information was used to show that there is a lateral interaction between each positively charged (+)N(CH(3))(3) headgroup and negatively charged PO(2)(-) headgroup of the adjacent DPPC molecule in the adsorbed vesicles and LB films. This study provides a framework for the use of this IR technique in studies of adsorption and transport of molecules across membrane interfaces.

Small diamines as modifiers for phosphatidylcholine/phosphatidylserine coatings in capillary electrochromatography

Greater stability of liposome coatings and improved resolution of model steroids in capillary electrochromatography (CEC) were sought by adding small diamines (ethylenediamine, diaminopropane, bis-tris-propane, or N-(2-hydroxyethyl)piperazine- N′-(2-ethanesulfonic acid, HEPES)) to the liposome solution before coating of fused silica capillaries. The phospholipid coatings consisted of 1 mM of 8:2 mol% phosphatidylcholine (PC)/phosphatidylserine (PS) and 5 mM of modifier in buffer solutions (acetate, phosphate, or Tris) at pH 4.0–7.4. The coating was based on a published procedure, and five steroids were used as neutral model analytes in evaluation of the coating. The results showed that under optimal conditions, the small linear diamines increased the packing density of anionic phospholipids, leading to improved separations. In addition, the choice of buffer for the liposome coating and separation appeared to influence the performance of the coatings. While buffers with amino groups take part in the phospholipid bilayer formation, buffers like phosphate may even have negative effect on coating formation. The factors affecting phospholipid coatings with diamines as modifiers are clarified.

Photo-induced proton gradients and ATP biosynthesis produced by vesicles encapsulated in a silica matrix

Sol-gel immobilization of soluble proteins has proven to be a viable method for stabilizing a wide variety of proteins in transparent inorganic matrices. The encapsulation of membrane-bound proteins has received much less attention, although work in this area suggests potential opportunities in microarray technology and high-throughput drug screening. The present paper describes a liposome/sol-gel architecture in which the liposome provides membrane structure and protein orientation to two transmembrane proteins, bacteriorhodopsin (bR) and F(0)F(1)-ATP synthase; the sol-gel encapsulation converts the liposomal solution into a robust material without compromising the intrinsic activity of the incorporated proteins. Here we report on two different proteoliposome-doped gels (proteogels) whose properties are determined by the transmembrane proteins. Proteogels containing bR proteoliposomes exhibit a stable proton gradient when irradiated with visible light, whereas proteogels containing proteoliposomes with both bR and F(0)F(1)-ATP synthase couple the photo-induced proton gradient to the production of ATP. These results demonstrate that materials based on the liposome/sol-gel architecture are able to harness the properties of transmembrane proteins and enable a variety of applications, from power generation and energy storage to the powering of molecular motors, and represent a new technology for performing complex chemical synthesis in a solid-state matrix.

Induction of apoptosis of human lung carcinoma cells by hybrid liposomes containing polyoxyethylenedodecyl ether

Hybrid liposomes can be prepared by simply ultrasonicating a mixture of vesicular and micellar molecules in aqueous solution. A clear solution of hybrid liposomes composed of 90 mol% dimyristoylphosphatidylcholine (DMPC) and 10 mol% polyoxyethylene(23)dodecyl ether (C 12(EO) 23) having a hydrodynamic diameter of 100–120 nm was obtained. Highly inhibitory effects of hybrid liposomes of 90 mol% DMPC/10 mol% C 12(EO) 23 on the growth of human lung carcinoma (RERF-LC-OK and A549) cells without any drugs were obtained. Induction of apoptosis by hybrid liposomes in RERF-LC-OK and A549 cells was verified on the basis of fluorescence microscopy, agarose gel electrophoresis of DNA and flow cytometry. We elucidated the pathway of apoptosis induced by hybrid liposomes as follows: (a) accumulation of hybrid liposomes in tumor cell membrane was revealed using microphysiometer. (b) Reduction of mitochodrial membrane potential and activation of caspase-9, -3 and -8 were obtained, indicating that apoptotic signal by hybrid liposomes should pass through mitochondria and these caspases. It is worthy to note that such a novel mechanism of apoptosis induced by hybrid liposomes without any drugs was performed for the first time in human lung carcinoma cells.

Influence of pH on formation and stability of phosphatidylcholine/phosphatidylserine coatings in fused-silica capillaries

The effect of pH on the formation and stability of phospholipid coatings in fused-silica capillaries in electrophoresis was investigated. A liposome solution consisting of 3 mM of 80:20 mol% phosphatidylcholine/phosphatidylserine (PC/PS) in N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) buffer was used as coating material. The coating was prepared by a method described earlier and five steroids were used as neutral model analytes. First, the effect of pH of the coating solution on the formation and stability of phospholipid coatings was studied at pH 6.5-8.5. The pH of the background electrolyte (BGE) solution (HEPES) was either kept constant at pH 7.4 or made similar to the pH of the liposome coating solution. Results showed that attachment of the coating on the fused-silica wall mostly depends on the protonation of amines of the phospholipids and HEPES. The ability of the phospholipid coating to withstand changes in pH was then investigated by coating at pH 7.5 and separating steroids with acetic acid, 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), HEPES, or glycine BGE, adjusted to pH between 4.5 and 10.8. The results showed that with use of BGE solution at pH 10.8, the separation of steroids was not successful and the electroosmotic flow was high because of leakage of the phospholipid coating during preconditioning of the capillary with BGE solution. There was no phospholipid leakage with a BGE solution of pH 4.5, indicating that the protonated form of the functional groups of PS and HEPES participating in the attachment of the phospholipid coating to the capillary play an essential role in the success of the coating.

Pharmacokinetic and phototherapeutic studies of monocationic methoxyphenylporphyrin derivative

Photodynamic therapy, a novel treatment for cancer, works through photoactivation of a tumor-localized photosensitive drug, and localized through oxidative damage to kill cells and ablate tumors. Pharmacokinetic and phototherapeutic properties of a cationic porphyrin were assayed in a Balb/c mouse cancer model in order to evaluate its efficiency as photosensitizer. Biodistribution studies were carried out by intraperitoneal injection of 5mg/kg CP incorporated into a liposome solution. CP was recovered from serum and organs at various times after treatment. The serum biochemical parameters and histological studies were used to test hepatic and renal functionality. For phototherapeutic studies, the light source used was a slide projector (360J/cm(2)). The efficiency of CP was evaluated by following tumor growth curves for 10 days after PDT doses. Immunohistochemical detection was carried out to evaluate caspase-3 activation in CP-PDT-treated tumors. The photosensitizer distribution suggests that CP is mainly eliminated from the organism via the bile-gut pathway, and that neurotoxic and cutaneous photosensitivity effects are reduced or absent. The porphyrin distribution from bloodstream to tissue began at 24h of drug administration. CP did not affect the hepatic and renal functionality, as was demonstrated by the physiological parameters. PDT-treated tumors showed delay in growth rate as compared to untreated control mice. Biochemical studies showed that the efficient tumor regression is dependent on caspase-3 activity signaling response associated with apoptosis. The results obtained suggest that the porphyrin CP may be a promising candidate for further use in PDT treatments.

Morphological change of a liposome induced by an oligonucleotide tagged with lipophilic unit

We synthesized a conjugate molecule which consisted of DNA, cholesterol, and poly(ethylene glycol) as a spacer. When DNA duplex containing this simple conjugate was added to a liposome solution, interestingly, a pearling transformation of the liposome was observed. Such a morphological change of the liposome is required in the study of synthesizing an artificial cellular life, because the cell division is one of the essential characteristic of the life system.

Structure of solid-supported lipid–DNA–metal complexes investigated by energy dispersive X-ray diffraction

When mixing aqueous solutions of DNA and dioloeyl-phosphatidylcholine multilamellar liposomes in the presence of Mn 2+ ions, a multilamellar liquid-crystalline phase is formed in which DNA monolayers are comprised between opposing lipid bilayers. Here, we report on a new experimental procedure resulting in the formation of solid-supported DOPC–DNA–Mn 2+ complexes in the biologically relevant excess water condition. The supramolecular structure of solid-supported complexes, characterized by means of Energy Dispersive X-ray Diffraction, exhibits very similar structural properties with respect to the solution structure of the complexes as revealed by synchrotron Small Angle X-ray Scattering measurements.

Influence of cetyltrimethylammonium bromide on phosphatidylcholine-coated capillaries.

Large unilamellar vesicles of egg-phosphatidylcholine (eggPC), a naturally occurring phospholipid, were used in capillary electrophoresis (CE) for semi-permanent coating of fused silica capillaries. The stability of the phospholipid coating was tested at different cetyltrimethylammonium bromide (CTAB) concentrations with and without CaCl(2) present in the coating solution. The effect of physical factors influencing the coating stability (e.g. duration of the coating time, storage temperature of the coating solution) were also studied. Standing overnight in background electrolyte (BGE) solution did not alter the eggPC phospholipid coating noticeably. The performance of the coating was tested with a mixture of basic proteins (lysozyme, ribonuclease A and alpha-chymotrypsinogen A). Highest efficiencies (over 200,000 plates m(-1)) were achieved when the capillary was filled for 15 h with a liposome solution containing both CTAB and CaCl(2).

Attenuated total reflectance infrared studies of liposome adsorption at the solid–liquid interface

Interfacial interactions between liposomes and the solid–liquid interface (i.e. a ZnSe internal reflection element, modified to mimic a biological surface) were studied by Fourier transform infrared (FTIR) spectroscopy in attenuated total reflectance (ATR) mode. Both conventional liposomes, containing lecithin and cholesterol and Stealth ® liposomes containing poly(ethylene)glycol (PEG)5000- or PEG2000-lipids were investigated. IR bands due to the liposome components were observed to increase with time and enabled the liposome adsorption kinetics and thermodynamics to be quantified. The liposome solution conditions, surface properties and compositions have all been shown to influence liposome adsorption. Free energies of adsorption were determined to be in the range from −10.0 to −11.0 kJ mol −1 and slightly reduced by PEG incorporation. The adsorption rate constant is decreased with increased solution pH and decreased ionic strength; this reflects the importance of electrostatics in controlling liposome adsorption. Increasing the level and molecular weight of PEG incorporation in the liposomes significantly reduced both the rate and extent of liposome adsorption; steric hindrance is considered to play a key role. Findings from this research will improve the understanding of liposome interaction during drug delivery, give insight into the actions of liposomes in the body and may form the basis for improved liposome formulations.