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.     

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.     

Active uptake of drugs into photosensitive liposomes and rapid release on UV photolysis

Liposomes containing high concentrations of the anticancer drug doxorubicin, prepared by active-loading techniques, have been intensively investigated as potential agents for chemotherapy. The present study investigates the possibility of active uptake and photoinduced release of such solutes from liposomes incorporating a photoisomerizable lipid. The active loading of acridine orange and doxorubicin was investigated using liposomes containing entrapped ammonium sulfate. The liposomes were prepared with dipalmitoyl-L-alpha-phosphatidyl choline (DPPC) and a photochromic lipid, (1,2-(4'-n-butylphenyl)azo-4'-(gamma-phenylbutyroyl))-glycero-3- phosphocholine (Bis-Azo PC), which isomerizes on exposure to near-UV light with resulting changes in membrane permeability to solutes. The rate of loading of the vesicles below the phase transition temperature of DPPC was investigated as a function of Bis-Azo PC and cholesterol concentrations in the liposome. The rate of doxorubicin uptake was found to be greatly decreased in the presence of cholesterol, while below 30 degrees C the rate of acridine orange uptake was increased in the presence of cholesterol. On exposure to a single UV laser pulse, actively loaded acridine orange was rapidly released from liposomes containing Bis-Azo PC at a rate similar to that found for the indicator dye calcein. However while cholesterol had previously been shown to greatly enhance the rate of photo-induced calcein leakage, it had no significant effect on the rate of acridine orange release. After active loading into DPPC vesicles containing Bis-Azo PC, doxorubicin was also released after exposure to a single laser pulse, but at a rate slower than for acridine orange and calcein. The difference in behavior between these systems is ascribed to the interactions of acridine orange and doxorubicin with the liposome bilayer. Photoinduced release of pharmacologically active materials from sensitized liposomes might provide a useful adjunct or alternative to conventional photodynamic therapy.     

Electric impedance method for evaluation of the release property of calcein-encapsulated liposomes

This paper is concerned with the study on development of a novel method for evaluation of the liposomes release property by measuring the electric impedance changes of liposome suspensions. Calcein/NaOH encapsulated liposomes (calcein-liposomes) were prepared with deionized water and were treated with ultrasonic irradiation in order to investigate the release property of the liposomes. To validate the proposed impedance measuring method, the calcein release rates were evaluated both by the impedance changes and the fluorescence intensity changes in calcein-liposome suspensions. With the comparison of these results obtained by the two methods, it is shown that the impedance method has much wider detecting concentration range than the fluorescence one. Furthermore, the impedance method can be efficiently used for evaluation of the release property on various ionic substances encapsulated within liposomes.     

Water adsorption in ion-bearing nanopores

Grand canonical Monte Carlo simulations are used to examine the adsorption of water into cylindrical nanopores containing single ions. The isotherms for water adsorbing into nanopores with radii of 0.44, 0.54, 0.64, and 0.74 nm and containing Na+, K+, Ca2+, Cl-, or F- at 298 K are computed. In all cases the nanopores are found to fill at reservoir chemical potentials below the chemical potential of saturated water vapor at 298 K. The threshold chemical potential is found to be sensitive to both the size of the channel and the ion species, with the anion-bearing pores filling at lower chemical potentials. Additionally, the filling threshold chemical potential is found to decrease as the radius of the pores is decreased. Pores with K+ and Cl- are compared, and the Cl- pores are found to exhibit higher water densities in the filled states and a more energetically favorable water structure while yielding lower per particle entropies. Sample simulation configurations are also examined and indicate that at low chemical potentials, the adsorbed water forms a cluster around the ion. Finally, the influence of the choice of water model on the adsorption isotherms is examined.     

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.     

Studying the cavities in liquid ethyleneglycol

Liquid ethyleneglycol models of 1000 molecules in a simple cell shaped as a rectangular parallelepiped are constructed using molecular dynamics at temperatures of 268 to 443 K and a system pressure maintained close to zero. The cavities (pores) in ethyleneglycol and the time and temperature dependence of their sizes are analyzed. The pores are found to arise in fluctuation in different areas of the model and to collapse rather quickly without exhibiting any dynamic connection. The radii of the largest pores are shown to grow from 1.674 to 2.174 Å formed predominantly by hydrogen and carbon atoms and appreciably depleted of oxygen atoms. It is established that the largest pores could easily accommodate argon atoms at temperatures above 385 K. The solubility of argon in ethyleneglycol is estimated as 0.1 mol % at 403 K and 0.5 mol % at 423 and 443 K.     

A new type of pH-sensitive phospholipid

We describe the synthesis and characterization of a type of pH-sensitive pentaerythritol phospholipids, using a trialkoxybenzylidene acetal as the acid-labile moiety. This lipid was prepared by an eight-step synthesis via a latentiation strategy. Liposomes were prepared via the thin film extrusion method. The changes of liposomal sizes were measured by dynamic light scattering. Content release rates of the liposomes as a function of pH were monitored by using a calcein fluorescence dequenching assay. These results indicated that this new liposomal system was capable of releasing its contents under mildly acidic conditions. At last, in vitro cytotoxicity was assayed against three cell lines, suggesting this type of phospholipids was low toxic.     

Effect of lipid composition on phospholipase A2-catalyzed membrane leakage in immobilized liposomes: sensitization for polychlorinated biphenyls detection with antibody affinity column tandem with fluorescent liposome column

Phospholipase A(2) (PLA(2))-catalyzed membrane leakage can be detected by immobilized liposomes containing a self-quenching fluorescent dye, calcein, on an open column using off-line analysis with a fluorescent spectrophotometer. The calcein release was found to be affected by the pH value, incubation time, and liposome compositions. The fluorescent signal from the negatively charged liposomes hydrolyzed by PLA(2) was 5 times higher than that from neutral liposomes. We utilized this enzymatic reaction to amplify signal to detect polychlorinated biphenyls (PCBs). To achieve this goal, we conjugated an analogue of PCB, 3,4-dichloroaniline, to PLA(2). The competitive immunoreaction between the 3,4-dichloroaniline-PLA(2) conjugate and PCB samples on the anti-PCB antibody column caused the release of the bound PLA(2) conjugates in proportion to the PCB concentration. The released PLA(2) conjugates was then passed through the tandem fluorescent liposome column causing release of fluorescent dye from the liposomes. Therefore, the signal of immunocompetitive assay was amplified on the fluorescent liposome column. The tandem column system achieves a high sensitivity by detecting the PCB concentration as low as 0.5 ng/mL in less than 20 min. It has great potential in detecting other pollutants, and has been used for sensitive immunoassays.     

Amphotericin B dimers with bisamide linkage bearing powerful membrane-permeabilizing activity

[structure: see text] Covalently linked dimers of amphotericin B were prepared by cross-linking its carboxylic acid. Among these, a dimer with a linkage of 1,6-hexanediamine revealed potent hemolytic activity (EC50, 0.25 microM) while its N-acetyl derivative gave rise to large K+ ion flux in phosphatidylcholine liposomes, regardless of the presence or absence of sterols, suggesting that the dimers may serve as a tool for elucidating the structure of the ion channel assemblage formed by amphotericin B.     

Study of the interaction between cyclodextrins and liposome membranes: effect on the permeability of liposomes

We wanted to compare and understand the effect of the most currently used cyclodextrins on a model membrane. We studied the influence of most currently used cyclodextrins on the release of a fluorescent marker encapsulated in the inner cavity of SUV liposomes. It was shown that the observed effect on calcein release can be directly related to the affinity of cyclodextrins for both lipid components of liposomes, cholesterol and phosphatidylcholine. From this relationship, we were able to determine, for each cyclodextrin, a theoretical concentration giving rise to 50% or 100% calcein release. This theoretical concentration was confirmed experimentally.     

Design of liposomes containing photopolymerizable phospholipids for triggered release of contents

We describe a novel class of light-triggerable liposomes prepared from a photo-polymerizable phospholipid DC_8,9PC (1,2-bis (tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine) and DPPC (1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine). Exposure to UV (254 nm) radiation for 0–45 min at 25 °C resulted in photo-polymerization of DC_8,9PC in these liposomes and the release of an encapsulated fluorescent dye (calcein). Kinetics and extents of calcein release correlated with mol% of DC_8,9PC in the liposomes. Photopolymerization and calcein release occurred only from DPPC/DC_8,9PC but not from Egg PC/DC_8,9PC liposomes. Our data indicate that phase separation and packing of polymerizable lipids in the liposome bilayer are major determinants of photo-activation and triggered contents release.     

Bacterial toxin-triggered drug release from gold nanoparticle-stabilized liposomes for the treatment of bacterial infection

We report a new approach to selectively deliver antimicrobials to the sites of bacterial infections by utilizing bacterial toxins to activate drug release from gold nanoparticle-stabilized phospholipid liposomes. The binding of chitosan-modified gold nanoparticles to the surface of liposomes can effectively prevent them from fusing with one another and from undesirable payload release in regular storage or physiological environments. However, once these protected liposomes "see" bacteria that secrete toxins, the toxins will insert into the liposome membranes and form pores, through which the encapsulated therapeutic agents are released. The released drugs subsequently impose antimicrobial effects on the toxin-secreting bacteria. Using methicillin-resistant Staphylococcus aureus (MRSA) as a model bacterium and vancomycin as a model anti-MRSA antibiotic, we demonstrate that the synthesized gold nanoparticle-stabilized liposomes can completely release the encapsulated vancomycin within 24 h in the presence of MRSA bacteria and lead to inhibition of MRSA growth as effective as an equal amount of vancomycin-loaded liposomes (without nanoparticle stabilizers) and free vancomycin. This bacterial toxin enabled drug release from nanoparticle-stabilized liposomes provides a new, safe, and effective approach for the treatment of bacterial infections. This technique can be broadly applied to treat a variety of infections caused by bacteria that secrete pore-forming toxins.     

Preparation and characterization of complexes of liposomes with gold nanoparticles

Gold nanoparticles (Au NPs), which are extremely useful materials for imaging and photothermal therapy, typically require a drug delivery system to transport them to the affected tissue and into the cells. Since liposomes are approved as drug carriers, complexes of liposomes with Au NPs were considered ideal solutions to deliver Au NPs to the target site in vivo. In this study, we prepared complexes of various liposomes with Au NPs via physical absorption and characterized them. The time dependency of the surface plasmon resonance of this complex, which is a unique property of Au NPs, shows that the liposomes promote the formation of stable dispersions of Au NPs under isotonic conditions, even though intact Au NPs aggregate immediately. From a release assay of calcein from liposomes and transmission electron microscopy analysis, the Au NPs were complexed with liposomes without membrane disruption. These complexes could be formed by using cationic liposomes and polyethylene glycol-modified liposomes, as well as by using phosphatidylcholine liposomes, which are useful for drug and gene delivery. We proposed this kind of complex as a nanomedicine with diagnostic and therapeutic ability.     

Colon targeted delivery systems of metronidazole based on osmotic technology: development and evaluation

Colon targeted delivery systems of metronidazole (MTZ) based on osmotic technology were developed. The developed systems consisted of osmotic core (drug, osmotic agent and wicking agent), coated with semipermeable membrane (SPM) containing guar gum as pore former, coated core were then further coated with enteric coating to protect the system from acidic environment of stomach. The effect of various formulation variables namely the level of wicking agent (sodium lauryl sulphate), osmotic agent in the osmotic core, the level of pore former (guar gum) in SPM, and the thickness of SPM, were studied on physical parameters and drug release characteristics of developed formulations. MTZ release was inversely proportional to SPM thickness, but directly related to the level of pore former, wicking agent and osmotic agent. On the other hand burst strength of the exhausted shells was decreased with the increase in level of pore former in the membrane but increased with the increase in the thickness of SPM. The drug release from the developed formulations was independent of pH, and agitation intensity, but dependent on the osmotic pressure of the release media. The thickness of enteric coating could prevent formation of delivery pores before contact with simulated colonic fluid, but had no effect on drug release. Result of SEM studies showed the formation of in-situ delivery pores in the membrane from where the drug release occurred, and the number of pores formed were directly related to the initial level of pore former (guar gum) in SPM. The manufacturing procedure was found to be reproducible and formulations were found to be stable during 3 months of accelerated stability studies.     

Tripolyphosphate-sensitive egg phosphatidylcholine liposomes incorporating hydrophobically modified poly(ethylene imine)

Liposomes, which release their contents in answer to tripolyphosphate (TPP, a penta-anion), were prepared by immobilizing hydrophobically modified poly(ethylene imine) (HmPEI) on the surface of egg phosphatidylcholine (egg PC) liposome. HmPEI was prepared by covalently attaching decanoyl chloride to PEI through a condensation reaction. According to the ¹H NMR spectrum, the number of decanoyl chloride per one molecule of PEI was about 21, and HmPEI was air/water interface-active. HmPEI could readily complex with TPP in HEPES buffer (30 mM, pH 7.0), confirmed by Fourier transformed infrared spectrophotometer spectroscopy. The complexation increased with increasing the concentration of HmPEI and TPP, investigated through the measurement of optical density and light scattering intensity. Liposomes incorporating HmPEI were prepared by a film hydration and sonication method. The liposomes were multi-lamellar vesicles, observed on transmission electron microscope. Liposomes free of HmPEI did not release calcien when they were mixed with TPP. Liposomes whose egg PC/HmPEI was relatively low (e.g., 20:1 and 20:2) released calcein but not extensively (less than 10%) when mixed with TPP. Liposomes whose egg PC/HmPEI was relatively high (e.g., 20:4 and 20:20) released calcein extensively. For example, when the liposomes with lager amount of HmPEI were mixed with TPP so that HmPEI/TPP weight ratio was 8:1, the release degree in 60 sec was more than 70%. HmPEI can complex with TPP through electrostatic interaction and the complexation was thought to cause perturbation in the liposomal membranes and trigger the release.     

Factors affecting physicochemical properties of liposomes prepared with hydrogenated purified egg yolk lecithins by the microencapsulation vesicle method

We examined hydrogenated purified egg yolk lecithins, having practical advantages over non-hydrogenated ones, as liposomal membrane materials. Liposomes were prepared by the microencapsulation vesicle (MCV) method in which liposomes are formed through two-step emulsification and dispersion. Three types of purified egg yolk lecithins with different iodine values were examined after being dissolved in one of three lipid solvents. The liposome size increased as the temperature during the second emulsification increased, being closer to the boiling temperature of the solvent. The preparation temperature in relation to the transition temperature of each lecithin was also a factor affecting liposome sizes. As for the encapsulation efficiencies of the model compound calcein in liposomes, they differed mainly depending on the solubility of each lecithin in a lipid solvent and it was more obvious in hydrogenated lecithins. A high preparation temperature resulted in lower encapsulation efficiencies, suggesting that leakage of encapsulated calcein was facilitated at high temperature in the MCV methods. There was a significant correlation between liposome sizes and encapsulation efficiencies in non-hydrogenated purified egg yolk lecithin but not in hydrogenated ones. When using hydrogenated purified egg yolk lecithins as liposomal membrane materials, it was suggested that a lipid solvent should be chosen so that a lecithin completely dissolves under the preparation condition in order to achieve a higher encapsulation efficiency. Smaller liposome particles were obtained when the second emulsification was performed at a lower temperature compared with the boiling point of the lipid solvent. These findings can be applied to control encapsulation efficiencies and particle sizes in each particular liposome preparation enclosing therapeutic agents.     

Capillary electrophoresis of anionic analytes in methanol: effect of counter-ions on electrophoretic mobility

Mobilities of 11 substituted benzoates and 3 nitrophenolates were determined in non-aqueous methanol with Li+, Na+, K+, Rb+, and tetrabutylammonium (Bu4N+) as counter-ions of the background electrolyte. The influence of the ionic concentration of the background electrolyte on the mobility of the analyte anions is more pronounced compared to aqueous solutions. The deviation from the dependence of the mobilities on the ionic strength from the Debye-Hückel-Onsager theory indicates the occurrence of ion-pair formation. For a given ion concentration (10 mmol/L), the decrease of the analyte mobility follows the counter-ion sequence Li+ < Na+ < K+ < Rb+, which is the inverse order of their Stokes radii. Bu4N+ as counter-ion has a similar effect on the analyte mobility than Li+ (which has the same Stokes radius, but a six times smaller crystal radius). Exceptions are some di- and trihydroxybenzoates. The mobilities in methanol and in water with the same counter-ion (Na+) at a given ionic concentration show very low correlation.     

Synthesis and vesicle formation of a nido-carborane cluster lipid for boron neutron capture therapy

The nido-carborane lipid, which has a double-tailed moiety, was synthesized from heptadecanol in 5 steps. Analysis in a transmission electron microscope by negative staining with uranyl acetate showed that the lipid formed a stable vesicle in which calcein was encapsulated. The lipid was incorporated into distearoylphosphatidylcholine (DSPC) liposomes at a very high concentration.     

Energetics of galactose, proline, and glutamine transport in a cytochrome-deficient mutant of Salmonella typhimurium.

The effect of inhibitors and uncouplers on the osmotic shock-sensitive transport systems for glutamine and galactose (by the beta-methyl galactoside permease) was compared to their effect on the osmotic shock-resistant proline and galactose permease systems in cytochrome-deficient cells of Salmonella typhimurium SASY28. Both osmotic shock-sensitive and -resistant systems were sensitive to uncouplers and to inhibitors of the membrane-bound Ca2+, Mg2+-activated adenosine triphosphatase. This suggests that uptake by both types of systems is energized in these cells by an electrochemical gradient of protons formed by ATP hydrolysis through the ATPase.