Detergent Removal Method Research Articles

Methods for obtaining phospholipid vesicles to improve the bioavailability of sparingly soluble active pharmaceutical ingredients

Low bioavailability of practically insoluble pharmaceutical substances is a serious issue in the field of drug development. The article provides an overview of the main methods for preparing liposomal drugs, such as the lipid film hydration method, ultrasonication method, extrusion method, high pressure homogenization method, solvent injection method, detergent removal method, and the reversephase evaporation method.

A Phospholipase-A Activity in Soluble Leishmania Antigens Causes Instability of Liposomes.

This study aimed to investigate the existence of phospholipase-A (PLA) activity in Soluble L. major Antigens (SLA) because of no reports for it so far. Liposomes were used as sensors to evaluate PLA activity. Liposomal SLA consisting of Egg Phosphatidylcholine (EPC) or Sphingomyelin (SM) were prepared by two different methods in different pH or temperatures and characterized by Dynamic Light Scattering (DLS) and Thin Layer Chromatography (TLC). Lipid hydrolysis led to the disruption of EPC liposomal SLA in both methods but the Film Method (FM) produced more stable liposomes than the Detergent Removal Method (DRM). The preparation of EPC liposomal SLA at pH 6 via FM protected liposomes from hydrolysis to some extent for a short time. EPC liposomes but not SM liposomes were disrupted in the presence of SLA. Therefore, a phospholipid without ester bond such as SM should be utilized in liposome formulations containing PLA as an encapsulating protein.

Liposomes Composed of Dioleoylphosphatidylethanolamine and 2-(Hexadecyloxy)Cinnamic Acid: Effects of UV Irradiation and pH Value on Release

Novel liposomes composed of dioleoylphosphatidylethanolamine (DOPE) and 2-(hexadecyloxy) cinnamic acid (HOCA) were prepared by a detergent removal method. When the molar ratio of DOPE to HOCA was 4:1, 3:2, and 2:3, the florescence quenching degree of 5(6)-carboxylic fluorescein (CF, a fluorescence marker) loaded in the assemblies was 62.4%, 68.9%, and 72.5%, respectively. The quenching degree was fairly high, indicating the assemblies were closed bilayer vesicles (liposomes). The release degree in 60 min of CF from the DOPE/HOCA liposomes was 9.6% to 15.7%, when the liposomes were subjected to the irradiation of a UV light (e.g., lambda = 254 nm). The photo-dimerization of the cinnamic acid residue of HOCA may destabilize the liposomal membrane, leading to a significant release. In addition, DOPE/HOCA (4/1) liposome exhibited a pH-sensitive release. It showed almost 100% of release degree in a few seconds under an acidic condition (e.g., pH 5.0), but it was stable at neutral and alkali conditions.

Non-Invasive Ophthalmic Liposomes for Nucleic Acid Delivery to Posterior Segment of Eye

Nucleic acids like siRNA and pDNA are remarkable for treatment of ophthalmic diseases in posterior segment of eye such as age-related macular degeneration (AMD). However, hydrophilic and high molecule compounds are restricted in intraocular distribution through anterior segment of the eye. In addition, the ocular tissue has a blood-retinal barrier which restricts drug delivery thorough systemic administration. Therefore the invasive intravitreal injection has been generally applied for treatment of retinal diseases. The objective in this study is to prepare nucleic acid-loaded liposomes for effective gene delivery to posterior segment of eye by non-invasive ophthalmic administration such as eye-drops. The pDNA/PEI-complex loaded liposomes were prepared using detergent removal method. The obtained liposomes were lyophilized with optimal amount of a cryoprotectant to avoid changes in physical properties and, followed by adjustment of an appropriate volume and osmotic pressure as ophthalmic solution. The liposomes show high pDNA encapsulation efficiency and good cellular uptake ability in human retinal pigment epithelial cells (ARPE-19 cells). We further demonstrate that the modification of ligand which binds to specific receptor on the RPE cells to the liposomes may improve gene delivery efficacy to the posterior segment of eye by non-invasive ocular instillation.

Detergent-Mediated Formation of Polymer-Supported Phospholipid Bilayers

Supported phospholipid bilayers can be formed by established methods such as vesicle fusion and the Langmuir-Blodgett (LB) technique. However, challenges remain in regards to creating supported bilayers from various lipid compositions, using various support surfaces, and incorporating membrane proteins. Here we report a detergent removal method as an alternative means of supported bilayer formation. The process consists of three steps: (1) incubation of phospholipid-poly(ethylene glycol) (PEG)-grafted glass with lipid-detergent micelles; (2) detergent removal by washing the surface with vesicles; and (3) incubation with the vesicles to complete lipid adsorption. These procedures yielded fluid planar bilayers of zwitterionic lipids. Because fluid structures were not obtained by vesicle fusion, the detergent seemed necessary to produce the polymer-supported bilayers. While anionic phospholipids inhibited the attachment of fluid bilayers in the absence of calcium ions, supported bilayers with almost full mobility were obtained from lipid mixtures containing 10-20 mol % anionic lipids in the presence of calcium ions. The incorporation of the anionic lipids in the bulk-facing leaflet was demonstrated by the binding of dye-labeled annexin V.

HDL superphospholipidation enhances key steps in reverse cholesterol transport

HDL-phospholipids (HDL-PL) play an important role in reverse cholesterol transport (RCT). Phosphatidylcholine (PC) is the most important phospholipid in RCT because it is the essential cholesterol-binding component of lipoproteins and is the acyl donor in the esterification of FC by lecithin:cholesterol acyltransferase (LCAT). FC efflux to sera is a positive anti-atherogenic function of HDL-PL. Although PC has long been recognized as an anti-atherogenic agent, development of new HDL therapies based on PC has been fraught with issues of efficacy, cost, and safety. Moreover, some methods to increase HDL-PC perturb HDL and release lipid-free apolipoproteins (apo) A-I. We developed a new method, HDL SPLn (SPLn) using a modified detergent removal method that obviates these concerns. SPLn can incorporate PC into HDL and increase HDL-PC > 10-fold. This is achieved with no loss of apo A-I. According to size exclusion chromatography and native gradient gel electrophoresis, SPLn raises the HDL particle weight in a dose-dependent way, from ∼120 to ∼350 kDa. Kinetic analysis of FC efflux to the resulting SPLn particles shows that K m and V max for SPLn HDL are lower and higher respectively than for native HDL. As a consequence, the catalytic efficiency, V max/ K m, increases by more than 400%. Clinically, small increases in serum HDL-PL are associated with significant and profound increases in FC efflux to serum. Treatment of relatively small amounts of plasma by SPLn is a potential method of improving at least one step in RCT.

Current trends in the production and biomedical applications of liposomes: a review

A review of literature was carried out to determine methods of production of liposomes, their stability, biodistribution and their uses as drug delivery systems. The conventional method of preparing liposomes is basically for the multilamellar vesicles (MLVs). However, other methods are used to reduce the size of these MLVs to small unilamellar vesicles (SUVs) so as to increase their plasma lifetime and consequently increase the possibility of achieving greater tissue localisation. Some of these methods of size reduction are sonication and high pressure extrusion. Each of these methods has its own advantages and disadvantages. Large unilamellar vesicles (LUVs), on the other hand, are prepared mainly by detergent removal method and reverse phase extrusion technique. There are also improved pharmacokinetic properties with liposomal drugs compared to free drugs, though some formulation factors affect the release kinetics of the liposomal drugs. The review also shows that liposomes have a lot of biomedical applications and uses. They have been used in drug targeting, oral delivery of vaccines, insulins, peptides and some compounds, which are usually degraded in the gastrointestinal tract. It has also found application in topical therapy especially in the eye and lungs. Other areas of application are in cancer chemotherapy and treatment of human immunovirus (HIV) infection. The control of the stability of liposomes is an essential pre-requisite for effective use as drug carriers. Leakage of the liposome is attributable mainly to differences in lamellar structure. For instance, MLVs are less prone to leakage than ULVs. The use of a combination of saturated phospholipid and cholesterol in the formulation of the liposomes has also been found to enhance stability with lower tendency to leakage. Keywords: Liposomes,phospholipid, encapsulation, applications Journal of Medicine and Biomedical Research Vol. 4 (1) 2005: pp. 9-21

Liposomes incorporating hydrophobically modified glucose oxidase

Glucose oxidase (GOD) was hydrophobically modified by conjugating palmitic acids to the amino groups of the enzyme. The number of palmitic acid residues conjugated to GOD was determined by using trinitrobenzenesulfonic acid (TNBS). According to the result, six palmitic acids turned out to be covalently attached to one GOD molecule. The activity of the modified GOD was 75%–80% of that of the native one, and due to the conjugated alkyl chain, the modified GOD was more surface-active than the native one. The modified GOD was incorporated into the liposomal bilayer of dioleoylphosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) by a detergent removal method. According to the results of the pH-dependent release, no significant release was observed in 60 sec at pH 8.2. An appreciable amount of calcein released at pH 7.0. A marked amount released at pH 6.0. The degree of release became more extensive at pH 5.0 and pH 5.6. This is possibly because the liposome is disintegrated into hexagonal phases under acidic conditions.

Glycosaminoglycan-resistant and pH-sensitive lipid-coated DNA complexes produced by detergent removal method

Cationic polymers are efficient gene delivery vectors in in vitro conditions, but these carriers can fail in vivo due to interactions with extracellular polyanions, i.e. glycosaminoglycans (GAG). The aim of this study was to develop a stable gene delivery vector that is activated at the acidic endosomal pH. Cationic DNA/PEI complexes were coated by 1,2-dioleylphosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) (3:2 mol/mol) using two coating methods: detergent removal and mixing with liposomes prepared by ethanol injection. Only detergent removal produced lipid-coated DNA complexes that were stable against GAGs, but were membrane active at low pH towards endosome mimicking liposomes. In relation to the low cellular uptake of the coated complexes, their transfection efficacy was relatively high. PEGylation of the coated complexes increased their cellular uptake but reduced the pH-sensitivity. Detergent removal was thus a superior method for the production of stable, but acid activatable, lipid-coated DNA complexes.

PH-dependent release property of dioleoylphosphatidyl ethanolamine liposomes

pH-sensitive liposomes were prepared by a detergent removal method. Dioleoylphosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) were combined for the preparation of the liposomes so that the molar ratios of DOPE to CHEMS are 9/1, 8/2, 6/4 and 5/5. On transmission electron micrographs, hundreds of nm sized-multilamella vesicles were observed. The degrees of fluorescence quenching were approximately 70–80%, indicating that closed vesicles were formed. According to the results of the pH-dependent release experiment with the liposome composed of DOPE/CHEMS (5/5), no significant release was observed in the pH region ranging from 6 to 8. At pH of 5, an appreciable amount of calcein was released. The patterns of pH-dependent releases from liposomes composed of DOPE/CHEMS (6/4) and DOPE/CHEMS (8/2) were almost the same as those from liposomes composed of DOPE/ CHEMS (5/5). With the liposomes composed of DOPE/CHEMS (9/1), unlike the other liposomes described above, almost 90% release was observed at pH 6. In this case, since the amount of a complementary molecule, CHEMS, is relatively small, the liposomes would be subjective to destabilization even at a small change in the degree of deionization of the carboxylic group. This may explain why the liposome of DOPE/CHEMS (9/1) exhibits a significant release at a relatively high pH, pH 6.0.

Effect of PEG lipid on fusion and fission of phospholipid vesicles in the process of freeze-thawing

The effects of freeze-thawing on the sizes and size distribution of egg yolk phosphatidylcholine (EggPC) liposomes in the presence of 0–40 mol% distearoylphosphatidylethanolamine–polyethyleneglycol 2000 (DSPE-PEG2000) prepared by detergent removal method were studied by quasielastic light scattering (QELS), gel exclusion chromatography, and freeze-fracture electron microscope. Especially, gel exclusion chromatography successfully provided the size distribution of polydisperse vesicle suspension. The mean diameters of the liposomes, which had originally large size, decreased with increase in the number of freeze-thawing cycles. On the contrary, the mean diameters of the liposomes, which had originally small size, increased with increase in the numbers of freeze-thawing cycles. After freeze-thawing over 10 times, the liposomal mean diameters fell into a range from 80 to 150 nm in spite of their original size. Gel exclusion chromatography showed that in the process of freeze-thawing of the liposomes containing DSPE-PEG 2000, two opponent events, one is fission and the other is fusion, occurred at the same time.

GTP/GDP exchange by Sec12p enables COPII vesicle bud formation on synthetic liposomes.

The generation of COPII vesicles from synthetic liposome membranes requires the minimum coat components Sar1p, Sec23/24p, Sec13/31p, and a nonhydrolyzable GTP analog such as GMP-PNP. However, in the presence of GTP and the full complement of coat subunits, nucleotide hydrolysis by Sar1p renders the coat insufficiently stable to sustain vesicle budding. In order to recapitulate a more authentic, GTP-dependent budding event, we introduced the Sar1p nucleotide exchange catalyst, Sec12p, and evaluated the dynamics of coat assembly and disassembly by light scattering and tryptophan fluorescence measurements. The catalytic, cytoplasmic domain of Sec12p (Sec12DeltaCp) activated Sar1p with a turnover 10-fold higher than the GAP activity of Sec23p stimulated by the full coat. COPII assembly was stabilized on liposomes incubated with Sec12DeltaCp and GTP. Numerous COPII budding profiles were visualized on membranes, whereas a parallel reaction conducted in the absence of Sec12DeltaCp produced no such profiles. We suggest that Sec12p participates actively in the growth of COPII vesicles by charging new Sar1p-GTP molecules that insert at the boundary between a bud and the surrounding endoplasmic reticulum membrane.

Effect of a PEG lipid (DSPE-PEG2000) and freeze-thawing process on phospholipid vesicle size and lamellarity

Liposomes containing distearoylphosphatidylethanolamine with covalently linked polyethylene glycol of molecular weight 2,000 (DSPE-PEG2000) covering a range of 0–30 mol% were prepared by a mechanical dispersion or detergent-removal method. The effects of DSPE-PEG2000 on particle sizes and lamellarity of liposomes were investigated. The average diameters of vesicles prepared from both methods decreased when the concentration of DSPE-PEG2000 was increased. The decrease in vesicle size with increase in DSPE-PEG2000 was ascribed to the steric hindrance of strongly hydrated PEG. The significant decrease in the sizes of DSPE-PEG2000-containing EggPC vesicles prepared by the detergent-removal method could be explained by the postvesiculation size growth in the process of micelle–vesicle transition. For DMPC vesicles prepared by the detergent-removal method, electron micrographs showed that inclusion of DSPE-PEG2000 promoted vesicle formation. Based on the results of investigation of calcein entrapment efficiency, we concluded that the lamellarity of liposomes is reduced as PEG lipid concentration is increased. Fragmentation of multilamellar vesicles into smaller unilamellar vesicles occurred more readily when the liposome suspension was subjected to repetitive freeze-thawing. After five cycles of freezing and thawing, vesicles containing more than 0.5 mol% DSPE-PEG2000 were fragmented into unilamellar vesicles with diameters smaller than 300 nm.

Lipid-coated polyplexes for targeted gene delivery to ovarian carcinoma cells.

A nonviral gene delivery vector has been developed in our laboratory based on the cationic polymer, poly(2-(dimethylethylamino)ethyl methacrylate) (p(DMAEMA)). p(DMAEMA)-based polyplexes have been successfully used for the transfection of OVCAR-3 cells in vitro. However, these polyplexes were unable to transfect OVCAR-3 cells growing in the peritoneal cavity of nude mice after intraperitoneal administration, which could be ascribed to inactivation by components (including hyaluronic acid) present in the tumor ascitic fluid. The present work aimed at (a) protecting p(DMAEMA)-based polyplexes against destabilization or inactivation by polyanions such as hyaluronic acid present in tumor ascitic fluid and (b) enhancing cellular uptake of the protected p(DMAEMA)-based polyplexes by targeting with antibody Fab' fragments. To fulfill these requirements, we have developed a detergent removal method to coat polyplexes with anionic lipids. With this method, spherical particles of approximately 125 nm, which were protected from destabilization by polyanions, were obtained. More importantly, the transfection efficiency of lipopolyplexes was unaffected in the presence of hyaluronic acid, indicating that lipid coating of polyplexes protects against destabilization by hyaluronic acid. By conjugating antibody Fab' fragments directed against the epithelial glycoprotein-2 to the lipidic surface of these lipopolyplexes, target cell-specific transfection of OVCAR-3 cells could be obtained in vitro.

Preparation of influenza virosome vaccine with muramyldipeptide derivative B30-MDP

An influenza virosome vaccine, consisting of influenza hemagglutinin-neur-aminidase antigens (HANA), the muramyldipeptide derivative 6-O-(2-tetra-decylhexadecanoyl)-N-acetyl-muramyl-L-alanyl-D-isoglutamine (B30-MDP) and cholesterol, was prepared by the detergent removal method using a flow-through dialyzer, Liposomat. The shape of the influenza virosome vaccine mimicked that of the influenza virus, namely, B30-MDP/cholesterol vesicle covered with HANA spikes. The particle size and size distribution of prepared virosomes were evaluated by quasi-elastic laser light scattering and gel permea tion chromatography. The state of incorporation of HANA spikes at the membrane surface of virosome was confirmed by electron microscopy. The results indicated that virosome formation was influenced by initial B30-MDP concentration in the mixed micellar solution prior to dialysis. It is apparent that the initial concentration of B30-MDP, relative to the mixed-micelle-to-viro-some transition, is an important factor for virosome preparation. Accordingly, the transition was evaluated and clarified by the changes in membrane fluidity and particle size during the dialysis process. The results indicated that it is important to adjust the initial B30-MDP concentration in the mixed micellar solution before dialysis so that the concentration for the mixed-micelle-to-vesicle transition coincides with that for mixed-micelle-to-rosette transition appropriate for virosome formation.

Characteristics of the Membrane Permeability of Temperature-Sensitive Liposome

Abstract The relationship between the temperature-sensitivity of the release of an entrapped drug from liposome as well as the type of drug or liposome is discussed. The drug release from DPPC liposome (dipalmitoylphosphatidylcholine liposome) has been confirmed to increase abruptly near its phase-transition temperature for both low-molecular-weight compouds (such as calcein) and high-molecular-weight compounds (such as dextran). The amount of temperature-sensitive calcein released from the liposomes was dependent on the method of liposome preparation: that is, the release from REV (reverse phase evaporated vesicle) was the highest; next was that from LUV (large unilamellar vesicle prepered by detergent removal method), and lowest was from SUV (small unilamellar vesicle prepared by an ultrasonication method). In order to examine the size effect of liposomes on temperature-sensitive drug release, REV was fractionated into three groups according to their average sizes and calcein release for each group was investigated. The temperature at which calcein release showed a maximum was almost the same in each of three groups; while as the average liposome size of a group was larger, the initial rate of calcein release became higher and the temperature at which calcein release began became lower. The amount of release of a entrapped drug was primarily dependent on the molecular weight of the drug, in spite of the variety of drugs investigated.

Preparation of asialofetuin-labeled liposomes with encapsulated human interferon-gamma and their uptake by isolated rat hepatocytes.

The selective delivery of human recombinant interferon (IFN)-gamma to isolated rat hepatocytes was studied with asialofetuin (AF)-labeled liposomes. AF-liposomes containing buffer solution were initially prepared by the detergent removal method, and IFN-gamma was subsequently encapsulated by the freeze-thawing method without loss of activity. Virtually no free [32P]IFN-gamma was internalized into isolated rat hepatocytes, whereas AF-liposomes containing [32P]IFN-gamma were taken up to a significant degree. Liposomal binding to the hepatocytes (estimated at 4 degrees C) was one-fifth of the uptake (estimated at 37 degrees C). Since the uptake was inhibited by the addition of free AF, AF-liposomes may be taken up by the action of galactose-binding protein on the hepatocytic cell surface. The liposome preparation method reported in this paper provides a useful means for the encapsulation of unstable macromolecules into AF-liposomes. AF-liposomes were found effectively to carry IFN-gamma into hepatocytes in vitro.

Partially reassembled high density lipoproteins. Effects on cholesterol flux, synthesis, and esterification in normal human skin fibroblasts.

The effects of a partially reassembled high density lipoprotein (PR-HDL) prepared by a detergent removal method on the cholesterol metabolism of fibroblasts has been tested under conditions of lipid depletion. The PR-HDL were composed of apolipoprotein A-I (1 mol) from human plasma HDL, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) 100 mol, and varying amounts of cholesterol. Below 15 mol%, the PR-HDL effected a net efflux of cholesterol from the fibroblasts; as a consequence, the activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase and acyl coenzyme A acyltransferase, respectively, were increased and decreased. Above 15 mol% cholesterol in the PR-HDL, net influx of cholesterol was observed and 3-hydroxy-3-methyl-glutaryl coenzyme A reductase activity was decreased, while acyl coenzyme A acyltransferase activity was increased. The uptake of several phosphatidylcholines, apo A-I, and cholesterol were measured as a function of time. The rates of lipid transfer to the cells decreased with increasing hydrophobicity of the transferring lipid. By contrast, in parallel experiments a relatively small quantity of 125I apo A-I was associated with the cells. Our results suggest that a component of lipid transfer from PR-HDL to fibroblasts occurs via monomeric transfer through the aqueous phase that is independent of apo A-I binding.