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 ...     

Liposomal Encapsulation of Carvacrol to Obtain Active Poly (Vinyl Alcohol) Films

Lecithins of different origins and compositions were used for the liposomal encapsulation of carvacrol within the framework of the development of active films for food packaging. Liposomes were incorporated into aqueous polymeric solutions from fully (F) and partially (P) hydrolysed Poly (vinyl alcohol) (PVA) to obtain the films by casting. The particle size distribution and ζ-potential of the liposomal suspensions, as well as their stability over time, were evaluated. Liposomal stability during film formation was analysed through the carvacrol retention in the dried film and the film microstructure. Subtle variations in the size distributions of liposomes from different lecithins were observed. However, the absolute values of the ζ-potential were higher (−52, −57 mV) for soy lecithin (SL) liposomes, followed by those of soy lecithin enriched with phosphatidylcholine (SL-PC) (−43, −50 mV) and sunflower lecithin (SFL) (−33, −38 mV). No significant changes in the liposomal properties were observed during the study period. Lyotropic mesomorphism of lipid associations and carvacrol leakage occurred to differing extents during the film drying step, depending on the membrane lipid composition and surface charge. Liposomes obtained with SL-PC were the most effective at maintaining the stability of carvacrol emulsion during film formation, which led to the greatest carvacrol retention in the films, whereas SFL gave rise to the least stable system and the highest carvacrol losses. P-PVA was less sensitive to the emulsion destabilisation due to its greater bonding capacity with carvacrol. Therefore, P-PVA with carvacrol-loaded SL-PC liposomes has great potential to produce active films for food packaging applications.     

Effect of water-soluble polymers on fusion of egg yolk and soybean phospholipid liposomes

The efficiencies of polyelectrolytes, i.e., polycations and polyanions, and several kinds of water-soluble polymers as fusogens on soybean phospholipid liposome (SL) and egg yolk phospholipid liposome (EL) were investigated by the fluorescence quenching method. There were optimal concentrations for the induction of fusion in every system. Polycations induced fusion of liposomes at very low concentration in comparison with other polymers. Poly(carboxylic acid)s induced fusion at relatively high concentration. A strong acidic polyanion with high molecular weight also induced fusion of liposomes. The induction efficiency of poly(ethylene glycol) on fusion was higher than other nonionic polymers. The efficiency of fusion of EL was lower than that of SL in all systems because of the higher stability of EL membrane. It was found that electrostatic interactions, hydrogen bonding and/or hydrophobic interaction between these water-soluble polymers and liposomal membranes played an important role on aggregation and fusion of liposomes.     

Glucose-triggered release from liposomes incorporating poly(<Emphasis Type="Italic">N</Emphasis>-isopropylacrylamide-co-methacrylic acid-co-octadecylacrylate) and glucose oxidase

In order to design liposomes which release their contents in a glucose-sensitive manner, the surfaces of egg phosphatidylcholine (egg PC) liposomes or dioleoylphosphatidylethanolamine (DOPE) liposomes were modified with the copolymer of N-isopropylacrylamide/methacrylic acid/octadecylacrylate and hydrophobically modified glucose oxidase (EC 1.1.3.4.). Whichever the liposomes were prepared with egg PC or DOPE, an extensive release of calcein was observed at acidic conditions. And DOPE liposomes were more pH sensitive than egg PC liposomes in terms of the release. In glucose-dependent calcein release experiment, there was no release for 180 min when the suspension of liposome was free of glucose. When the glucose concentration was 50 mg/dl, no appreciable amount of calcein was released for the first 50 min, but a significant release was observed for the last 130 min. At glucose concentration of 200 mg/dl, calcein release became more extensive and the releases for 180 min from egg PC and DOPE liposome were 84% and 98%, respectively.     

Destabilisation of liposomes by bovine serum albumin; Sepharose 2B-Cl experiment

Summary The stability of liposomes (2∶1 egg yolk lecithin:cholesterol, mole ratio; diameter about 100 nm) at increasing bovine serum albumin (BSA) concentration was study. The influence of introducing positive or negative charge to the liposomal bilayers was tested. The results indicated appreciable destructive effects of serum albumin on the liposomal membranes of neutral and negatively charged liposomes. Near-physiological concentration (30 mg mL⁻¹) of albumin dissolved more then 50% of liposomes. Presented at the 21^st ISC held in Stuttgart, Germany, 15th–20th September, 1996     

Liposomes Prepared from Inverse Micelles

A method for the preparation of liposome is introduced, which contains two experimental steps: (a) inverse micelles of lecithin are formed in water-in-oil system by sonication; (b) the micelles are spread on the water surface, passed Through the oil-water interface, and transformed into liposomes in the water phase. The main advantage of this method is that the inner aqueous solution encapsulated by liposomes could be different from their enviromental medium. The liposome size is less than 0.5 μm in diameter by atomic force microscope. Comparison of activities of urease with and without liposome encapsulation suggested that urease was well entraped into liposomes.     

Development of low cost pulmonary surfactants composed of a mixture of lipids or lipids-peptides using higher aliphatic alcohol or soy lecithin

The artificial pulmonary surfactant composition in the present study is characterized by a lipid mixture system composed of higher aliphatic alcohol, egg yolk phosphatidylcholine (egg PC), soy lecithin and higher aliphatic acid as the major components or a peptide-lipid mixture system composed of a combination of the lipid mixture system to which a peptide is added. Three peptides with amphiphilic surface-staying, membrane spanning, and both properties were designed and synthesized. The evaluation of pulmonary surfactant assay was performed by a hysteresis curve drawn upon the measurement for the surface tension-area curve with the Wilhelmy surface tensometer in vitro and the recovery of lung compliance for the pulmonary surfactant-deficient rat models in vivo. Lipid-mixture systems composed of octadecanol or soy lecithins containing no peptide were favorable hysteresis curves as compared with commercially available Surfacten((R)), but were not prominent. The peptide-lipid mixture systems composed of a combination of the lipid mixture of alkyl alcohol or soy lecithin to which peptides designed were added were desirable hysteresis curves similar to Surfacten((R)) and amphiphilic Hel 13-5 peptide-lipids mixture systems were much more effective than the lipid mixture system. Particularly, the recovery of lung compliance treated with hydrogenated soy lecithin-fractionated soy lecithin PC70-palmitic acid-peptide Hel 13-5 (40:40:17.5:2.5, w/w) was comparable to that with Surfacten((R)). Because the artificial pulmonary surfactant compositions of this study can be prepared at lower costs, they are useful for the treatment of respiratory distress syndrome and acute respiratory distress syndrome as well as for inflammatory pulmonary diseases, dyspnea caused by asthma, etc.     

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.     

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.     

Liposomal drugs dispersed in hydrogels. Effect of liposome, drug and gel properties on drug release kinetics

Release of calcein and griseofulvin (GRF) from control (gels in which solutes are dissolved in) and liposomal gels was studied using agarose-assisted immobilization as a technique to separate gels from drug-receptor compartments. Liposomes composed of phosphatidylcholine (PC) or distearoyl-glycero-PC and cholesterol (DSPC/Chol), and incorporating calcein or GRF were prepared by thin film hydration. After cleaning the liposomes they were dispersed in different hydrogels (carbopol 974 [1, 1.5 or 2% (w/w)], hydroxylethyl-cellulose (HEC) [4% (w/w)], or a mixture of the two), and release of calcein or GRF was followed by fluorescence or photometric technique, respectively. Results show that calcein release from liposomal gels is slower compared to control gels, and can be further retarded by using rigid-membrane liposomes (faster release from PC-liposome compared to DSPC/Chol-liposome gels). Additionally, calcein release is not affected by the lipid amount loaded (in the range from 2 to 8 mg/ml), therefore solute loading can be controlled according to needs.

Oppositely, GRF release from liposomal gels is determined by drug loading. At high drug loading levels (compared to GRF aqueous solubility), GRF is released with constant rate from liposomal gels irrespective of liposome type (PC or DSPC/Chol). Thereby, for amphiphilic/lipophilic drugs, drug properties (solubility, log P) determine the system behavior.

Calcein and GRF release from control carbopol gels is faster compared to HEC and mixture gels. The same is true for calcein in liposomal gels. Carbopol gel rheological properties were found to be significantly different (compared to the other gels), implying that these characteristics are important for drug diffusion from gels.     

Optimisation of preparation conditions and properties of phytosterol liposome-encapsulating nattokinase

Phytosterol liposomes were prepared using the thin film method and used to encapsulate nattokinase (NK). In order to obtain a high encapsulation efficiency within the liposome, an orthogonal experiment (L9 (3)(4)) was applied to optimise the preparation conditions. The molar ratio of lecithin to phytosterols, NK activity and mass ratio of mannite to lecithin were the main factors that influenced the encapsulation efficiency of the liposomes. Based on the results of a single-factor test, these three factors were chosen for this study. We determined the optimum extraction conditions to be as follows: a molar ratio of lecithin to phytosterol of 2?:?1, NK activity of 2500?U?mL?1 and a mass ratio of mannite to lecithin of 3?:?1. Under these optimised conditions, an encapsulation efficiency of 65.25% was achieved, which agreed closely with the predicted result. Moreover, the zeta potential, size distribution and microstructure of the liposomes prepared were measured, and we found that the zeta potential was -51?±?3?mV and the mean diameter was 194.1?nm. From the results of the scanning electron microscopy, we observed that the phytosterol liposomes were round and regular in shape and showed no aggregation.     

Preparation of curcumin-loaded liposomes and evaluation of their skin permeation and pharmacodynamics

This study aimed to investigate the in vitro skin permeation and in vivo antineoplastic effect of curcumin by using liposomes as the transdermal drug-delivery system. Soybean phospholipids (SPC), egg yolk phospholipids (EPC), and hydrogenated soybean phospholipids (HSPC) were selected for the preparation of different kinds of phospholipids composed of curcumin-loaded liposomes: C-SPC-L (curcumin-loaded SPC liposomes), C-EPC-L (curcumin-loaded EPC liposomes), and C-HSPC-L (curcumin-loaded HSPC liposomes). The physical properties of different lipsomes were investigated as follows: photon correlation spectroscopy revealed that the average particle sizes of the three types of curcumin-loaded liposomes were 82.37 ± 2.19 nm (C-SPC-L), 83.13 ± 4.89 nm (C-EPC-L), and 92.42 ± 4.56 nm (C-HSPC-L), respectively. The encapsulation efficiency values were found to be 82.32 ± 3.91%, 81.59 ± 2.38%, and 80.77 ± 4.12%, respectively. An in vitro skin penetration study indicated that C-SPC-L most significantly promoted drug permeation and deposition followed by C-EPC-L, C-HSPC-L, and curcumin solution. Moreover, C-SPC-L displayed the greatest ability of all loaded liposomes to inhibit the growth of B16BL6 melanoma cells. Therefore, the C-SPC-L were chosen for further pharmacodynamic evaluation. A significant effect on antimelanoma activity was observed with C-SPC-L, as compared to treatment with curcumin solution in vivo. These results suggest that C-SPC-L would be a promising transdermal carrier for curcumin in cancer treatment.     

Simultaneous measurements of K+ and calcein release from liposomes and the determination of pore size formed in a membrane.

The changes induced by biologically active substances in the permeability to K+ and calcein of liposomes composed of egg phosphatidylcholine and cholesterol were measured simultaneously in order to rapidly screen the sizes of pores formed in a membrane, using different sized markers. The substances examined in the present study were classified into three types based on differences in the rates at which K+ and calcein were released. The first type released only K+, and included gramicidin A. The second type predominantly released K+, preceding the release of calcein, and included amphotericin B and nystatin. The third type, including antimicrobial peptides, such as gramicidin S, alamethicin, and melittin, and several membrane-active drugs, like celecoxib (non-steroidal anti-inflammatory drug), 1-dodecylazacycloheptan-2-one (named azone; skin permeation enhancer), and chlorpromazine (tranquilizer), caused the release of K+ and calcein simultaneously. Thus, the sizes of pores formed in a liposomal membrane increased in the following order: types one, two, and three. We determined the size more precisely by conducting an osmotic protection experiment, measuring the release of calcein in the presence of osmotic protectants of different sizes. The radii of pores formed by the second type, amphotericin B and nystatin, were 0.36 - 0.46 nm, while the radii of pores formed by the third type were much larger, 0.63 - 0.67 nm or more. The permeability changes induced by substances of the third type are discussed in connection with a transient pore formed in a lipid packing mismatch taking place during the phase transition of dipalmitoylphosphatidylcholine liposomes.     

Deformable liposomes containing alkylcarbonates of γ-cyclodextrins for dermal applications

Liposomes made with hydrogenated soya lecithin (HPC) mixed with dodecylcarbonate γ-cyclodextrin (C12CD) at 20:1, 10:1 and 5:1 w/w ratios were prepared by the solvent evaporation method. C12CD had emulsifying properties and the possibility of producing deformable liposomes, as topical delivery system of progesterone (PG), was evaluated. Liposome size, deformability and drug entrapment were determined and the interaction between C12CD and HPC was investigated using differential scanning calorimetry (DSC). The size and the amount of PG loaded in the liposomes depended on the lipid:C12CD ratio: the smallest liposomes were obtained using 20:1 ratio and the maximum drug entrapment at 5:1 ratio. DSC analysis suggested that C12CD interacted with liposomes disrupting and fluidizing the lipid bilayer. PG transepidermal permeation through intact pig skin and PG skin uptake from deformable liposomes were assessed and compared to the values obtained from aqueous suspension and conventional liposomes. The PG permeations were negligible for all systems, while skin uptake increased for liposomes containing C12CD. This was attributed to the deformability and to the increase in the drug entrapment efficiency of these liposomes. The use of C12CD in liposome formulations can improve PG topical therapy.     

Liposome-templated supramolecular assembly of responsive alginate nanogels

Nanosized gel particles (nanogels) are of interest for a variety of applications, including drug delivery and single-molecule encapsulation. Here, we employ the cores of nanoscale liposomes as reaction vessels to template the assembly of calcium alginate nanogels. For our experiments, a liposome formulation with a high bilayer melting temperature (Tm) is selected, and sodium alginate is encapsulated in the liposomal core. The liposomes are then placed in an aqueous buffer containing calcium chloride, and the temperature is raised up to Tm. This allows permeation of Ca2+ ions through the bilayer and into the core, whereupon these ions gel the encapsulated alginate. Subsequently, the lipid bilayer covering the gelled core is removed by the addition of a detergent. The resulting alginate nanogels have a size distribution consistent with that of the template liposomes (ca. 120-200 nm), as confirmed by transmission electron microscopy and light scattering. Nanogels of different average sizes can be synthesized by varying the template dimensions, and the gel size can be further tuned after synthesis by the addition of monovalent salt to the solution.     

Covalent immobilization of liposomes on plasma functionalized metallic surfaces

A method was developed to functionalize biomedical metals with liposomes. The novelty of the method includes the plasma-functionalization of the metal surface with proper chemical groups to be used as anchor sites for the covalent immobilization of the liposomes. Stainless steel (SS-316) disks were processed in radiofrequency glow discharges fed with vapors of acrylic acid to coat them with thin adherent films characterized by surface carboxylic groups, where liposomes were covalently bound through the formation of amide bonds. For this, liposomes decorated with polyethylene glycol molecules bearing terminal amine-groups were prepared. After ensuring that the liposomes remain intact, under the conditions applying for immobilization; different attachment conditions were evaluated (incubation time, concentration of liposome dispersion) for optimization of the technique. Immobilization of calcein-entrapping liposomes was evaluated by monitoring the percent of calcein attached on the surfaces. Best results were obtained when liposome dispersions with 5mg/ml (liposomal lipid) concentration were incubated on each disk for 24h at 37°C. The method is proposed for developing drug-eluting biomedical materials or devices by using liposomes that have appropriate membrane compositions and are loaded with drugs or other bioactive agents.     

Integrity of liposomes in presence of various formulation excipients, when dispersed in aqueous media and in hydrogels

The integrity of liposomes when dispersed in presence of various common formulation excipients is studied. Additionally, the effect of the excipients on the release of calcein from the same liposomes when dispersed in hydrogels is investigated and the results of the two sets of experiments are compared. Propyleneglycol (PG), transcutol CG (TR), cremophor EL (CR) and labrafac hydro WL 1219 (LB) are used at 10 or 25% (v/v) and the retention of liposome encapsulated calcein is followed for 24 or 48 h periods. Calcein entrapping multilamellar liposomes composed of phosphatidylcholine (PC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) with or without addition of different amounts of cholesterol (Chol) were prepared by the thin film hydration method.

Experimental results reveal that liposomes are affected more by the excipients in the order: LB > CR > PG  TR. Particularly LB and in some cases also CR result in rapid release of most or the entire vesicle encapsulated dye. Addition of Chol in both PC and DSPC liposomes results in substantial increase of vesicle integrity in all cases. Concerning the release of calcein form the liposomal gels, from DSPC/Chol (1:1) liposomal gels calcein release was not affected by addition of 25% of TR or PG in all gels studied, but LB caused a significant increase in calcein release. However, from PC-liposomal gels even TR and PG (at 25%), increases calcein release.

Conclusively, the results of this study suggest that liposomes are protected from excipients when dispersed in gels compared to aqueous media. This should be taken into account when liposomal drug formulations are designed.     

Interaction of recombinant human interferon-gamma with liposomes

The interaction of recombinant human interferon-gamma (IFN) with egg phosphatidylcholine liposomes was studied. IFN which binds to liposomes was dependent on the liposomal charge and pH, and a preferential binding was observed in negatively charged liposomes at pH 7.4-10. Electron-microscopic observation showed that the increased liposomal turbidity induced by IFN was due to liposomal aggregation, and the increased turbidity could be decreased by the addition of NaCl. Thus, ionic binding may participate in this interaction. But, when the incubation time was longer, the liposomal aggregation was not decreased by the addition of NaCl, and the leakage of the entrapped marker, calcein, was observed. Electron-microscopic analysis showed that this leakage resulted from the morphological change of liposomes. From these findings, ionic binding may participate in the interaction between IFN and liposomes and then develop a morphological change in negatively charged liposomes under the neutral pH condition.     

Immobilization of liposomes on hydrophobically modified polymer gel particles in batch mode interaction

Immobilization of liposomal phospholipids onto Sephacryl S-1000 gels that were chemically conjugated with hydrophobic alkyl moieties, octyl, dodecyl and hexadecyl, was examined in batch mode interaction. Compared with the octyl gel, the dodecyl and the hexadecyl gels were found to immobilize the three to four times more phospholipids with the less hydrophobic moieties. The encapsulation of a water-soluble marker, with other evidences, suggests that the majority of the immobilized phospholipids maintained liposomal morphology. As the lipid of the interacting liposomes, egg yolk phosphatidylcholine (eggPC), 1,2-dimyristoylphosphatidylcholine (DMPC) and a mixture of DMPC and 1,2-dimyristamido-1,2-deoxyphosphatidylcholine were examined. At 22 °C, DMPC liposomes showed higher extent of immobilization than at 37 °C but not eggPC liposomes, suggesting that the phase of liposomal membrane could have influence on the immobilization. Exchange between the immobilized liposomes and free ones was found to be small, less than 3%. The gel that had been first interacted with liposomes to apparent saturation could further immobilize the newly added liposomes. The rate of this second immobilization was similar to that of the slow adsorption process; the both could be based on the same mechanism, possibly involving rearrangement of the immobilized liposomes on the gel as proposed by Lundahl. As had been observed in the flow mode, the immobilization had preference for smaller liposomes. In application of the system in batch mode, the size distributions of the immobilized liposomes and of those left in the supernatant may differ from that of the originally added liposomes.     

Structure and properties of complexes formed by cationic polymers and anionic cholesterol-containing liposomes

The adsorption of the synthetic polycation poly(N-ethyl-4-vinylpyridinium bromide) on the surface of three-component lipid vesicles (liposomes) formed from a mixture of anionic cardiolipin, electroneutral egg lecithin, and nonionic cholesterol is studied via laser microelectropheresis, dynamic light scattering, conductometry, fluorescence spectroscopy, and UV spectroscopy. The incorporation of cholesterol into the liposomal membrane increases its microviscosity; however, the membrane remains liquid-crystalline. Simultaneously, an increase in the fraction of cholesterol causes the formation of defects in liposome membranes during their binding with poly(N-ethyl-4-vinylpyridium bromide) and makes complexation irreversible. The results of this study are of interest for predicting the behavior of polyelectrolytes and biologically active structures formed on their basis on the surface of cells and the reaction of the cellular membrane to the adsorbed polymer.