Development of a Liposome Formulation for D-Cycloserine Local Delivery

Multilamellar liposomes loaded with D-cycloserine (D-CS) were prepared by a thin layer evaporation technique, followed by freezing and thawing cycles. Charged components and bioadhesive material, such as distearolylphosphatitylethanolamine covalently coupled with methoxypolyethyleneglycol, were used to prepare liposomes with different physico-chemical and technological properties. Negatively charged liposomes showed higher D-CS encapsulation efficiency (about 37%, w/w) than neutral and positively charged liposomes (about 5 and 17%, w/w, respectively). All formulations showed in vitro, after a burst effect, a prolonged release of the encapsulated drug.

Lipid vesicles made of dipalmitoylphosphatidylcholine (DPPC) were used as a biomembrane model to evaluate in vitro the interaction of D-CS with biological membranes. Differential scanning calorimetry was used as a simple and noninvasive technique of analysis. D-CS was distributed in the aqueous compartments of liposomes for interaction with the phospholipid polar head-groups (enhancement of Δ?H value). However, due to its high diffusibility the drug was also able to freely permeate through DPPC liposomes, altering during this passage the hydrophobic domains of the bilayers.

Stability studies were performed at different temperatures and pH values to assay the integrity of the drug during the liposome production steps. D-CS was rapidly degraded at acidic pH, but no significant hydrolysis was observed at pH 7.4 after 7 days.     

Encapsulation of Enrofloxacin in Liposomes I: Preparation and In Vitro Characterization of LUV

Liposomes are effectively used in the treatment of microbial infections. Higher cellular uptake has been reported when antibiotics are encapsulated in liposomes. In this study, enrofloxacin (ENF) was encapsulated in large unilamellar vesicles (LUVs) and the effects of formulation variables on the liposome characteristics were investigated. Liposomes were prepared using dry lipid film method. A number of variables such as molar ratios of phospholipid (DPPC; DL‐α‐phosphatidylcholine dipalmitoyl), cholesterol, ENF and amount of α‐tocopherol and the volumes of internal (chloroform) and external phases [phosphate buffered saline PBS (pH 7.4)] were studied. In vitro characterization of the liposomes including the encapsulation capacity, size and drug release properties were carried out. Using of this method, spherical LUV liposomes with high drug content could be produced. Particle size of liposomes changed between 3.12 and 4.95 µm. The molar ratios of DPPC, cholesterol and ENF affected the size of the liposome (p < 0.05). The drug encapsulation capacities were high and changed between 37.1% and 79.5%. The highest ENF encapsulation was obtained with the highest cholesterol content. An increase in the drug encapsulation capacity of the liposome was found with increasing molar ratios of DPPC, cholesterol and ENF (p < 0.05). Furthermore, the release of ENF from the liposomes decreased as the molar ratios of DPPC, cholesterol and ENF increased (p < 0.05). In conclusion, a convenient colloidal carrier for the controlled release of ENF can be prepared by changing the formulation parameters of LUVs.     

Encapsulation of enrofloxacin in liposomes I: preparation and in vitro characterization of LUV

Liposomes are effectively used in the treatment of microbial infections. Higher cellular uptake has been reported when antibiotics are encapsulated in liposomes. In this study, enrofloxacin (ENF) was encapsulated in large unilamellar vesicles (LUVs) and the effects of formulation variables on the liposome characteristics were investigated. Liposomes were prepared using dry lipid film method. A number of variables such as molar ratios of phospholipid (DPPC; DL-alpha-phosphatidylcholine dipalmitoyl), cholesterol, ENF and amount of alpha-tocopherol and the volumes of internal (chloroform) and external phases [phosphate buffered saline PBS (pH 7.4)] were studied. In vitro characterization of the liposomes including the encapsulation capacity, size and drug release properties were carried out. Using of this method, spherical LUV liposomes with high drug content could be produced. Particle size of liposomes changed between 3.12 and 4.95 microm. The molar ratios of DPPC, cholesterol and ENF affected the size of the liposome (p < 0.05). The drug encapsulation capacities were high and changed between 37.1% and 79.5%. The highest ENF encapsulation was obtained with the highest cholesterol content. An increase in the drug encapsulation capacity of the liposome was found with increasing molar ratios of DPPC, cholesterol and ENF (p < 0.05). Furthermore, the release of ENF from the liposomes decreased as the molar ratios of DPPC, cholesterol and ENF increased (p < 0.05). In conclusion, a convenient colloidal carrier for the controlled release of ENF can be prepared by changing the formulation parameters of LUVs.     

Spectroscopic and calorimetric studies on trazodone hydrochloride–phosphatidylcholine liposome interactions in the presence and absence of cholesterol

The interaction of antidepressant drug trazodone hydrochloride (TRZ) with dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposomes (MLVs) in the presence and absence of cholesterol (CHO) was investigated as a function of temperature by using Electron Paramagnetic Resonance (EPR) spin labeling, Fourier Transform Infrared (FTIR) Spectroscopy and Differential Scanning Calorimetry (DSC) techniques. These interactions were also examined for dimyristoyl phosphatidylcholine (DMPC) multilamellar liposomes by using Electron Paramagnetic Resonance (EPR) spin labeling technique. In the EPR spin labeling studies, 5- and 16-doxyl stearic acid (5-DS and 16-DS) spin labels were used to monitor the head group and alkyl chain region of phospholipids respectively. The results indicated that TRZ incorporation causes changes in the physical properties of PC liposomes by decreasing the main phase transition temperature, abolishing the pre-transition, broadening the phase transition profile, and disordering the system around the head group region. The interaction of TRZ with unilamellar (LUV) DPPC liposomes was also examined. The most pronounced effect of TRZ on DPPC LUVs was observed as the further decrease of main phase transition temperature in comparison with DPPC MLVs. The mentioned changes in lipid structure and dynamics caused by TRZ may modulate the biophysical activity of membrane associated receptors and in turn the pharmacological action of TRZ.     

Interaction of antimycobacterial and anti-pneumocystis drugs with phospholipid membranes

Liposomes can be used as carriers of drugs in the treatment of viral, bacterial and protozoal infections. The potential for liposome-mediated therapy of Mycobacterium avium-intracellulare complex infections, one of the most common opportunistic infections in AIDS, is currently under study. Here, we have investigated the effect of the lipid-soluble antimycobacterial drugs ansamycin, clofazimine and CGP7040 on the thermotropic behavior of liposomes composed of dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG) using differential scanning calorimetry (DSC). In the presence of ansamycin (rifabutine), the peak gel-liquid crystalline phase transition temperature (Tm) of DPPG was reduced, as was the sub-transition temperature (Ts), whereas the Tm of DPPC was reduced only slightly. The temperature of the pre-transition (Tp) of DPPC was lowered, while the pre-transition of DPPG was abolished. Ansamycin also caused the broadening of the transition endotherm of both lipids. Equilibration of the drug/lipid complex for 1 or 5 days produced different thermotropic behavior. In the presence of clofazimine, the cooperativity of the phase transition of DPPG decreased. Above 10 mol% clofazimine formed two complexes with DPPG, as indicated by two distinguishable peaks in DSC thermograms. The Tm of both peaks were lowered as the mole fraction increased. Clofazimine had minimal interaction with DPPC. In contrast, CGP7040 interacted more effectively with DPPC than with DPPG, causing a reduction of the size of the cooperative unit of DPPC even at 2 mol%. The main transition of DPPC split into 3 peaks at 5 mol% drug. The pre-transition was abolished at all drug concentrations and the sub-transition disappeared at 10 mol% CGP7040. These studies suggest that maximal encapsulation of clofazimine in liposomes would require a highly negatively charged membrane, while that of CGP7040 would necessitate a zwitterionic membrane. We have also investigated the interaction of the water-soluble antibiotic pentamidine, which has been used against Pneumocystis carinii, the most lethal of AIDS-related opportunistic pathogens. Aerosol administration of this drug leads to long-term sequestration of the drug in the lungs. The DPPG/pentamidine complex exhibited a pre-transition at 3.5 degrees C, an endothermic peak at 42 degrees C, and an exothermic peak at 44.5 degrees C, followed by another endothermic peak at 55 degrees C. The exotherm depended on the history of the sample, requiring pre-incubation for several minutes below the 42 degrees C transition. These observations suggest that upon melting of the DPPG chains at 42 degrees C, the DPPG crystallizes as a DPPG/pentamidine complex that melts at 55 degrees C.     

Chlorpromazine interaction with glycerophospholipid liposomes studied by magic angle spinning solid state (13)C-NMR and differential scanning calorimetry

Phosphatidylserine (PS) extracted from pig brain and synthetic dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were used to make DPPC/DMPC and DPPC/PS large unilamellar liposomes with a diameter of approximately 1 microm. Chlorpromazine-HCl (CPZ), an amphipathic cationic psychotropic drug of the phenothiazine group, is known to partition into lipid bilayer membranes of liposomes with partition coefficients depending on the acyl chain length and to alter the bilayer structure in a manner depending on the phospholipid headgroups. The effects of adding CPZ to these membranes were studied by differential scanning calorimetry and proton cross polarization solid state magic angle spinning (13)C-nuclear magnetic resonance spectroscopy (CP-MAS-(13)C-NMR). CP-MAS-(13)C-NMR spectra of the DPPC (60%)/DMPC (40%) and the DPPC (54%)/DMPC (36%)/CPZ (10%) liposomes, show that CPZ has low or no interaction with the phospholipids of this neutral and densely packed bilayer. Conversely, the DPPC (54%)/PS (36%)/CPZ (10%) bilayer at 25 degrees C demonstrates interaction of CPZ with the phospholipid headgroups (PS). This CPZ interaction causes about 30% of the acyl chains to enter the gauche conformation with low or no CPZ interdigitation among the acyl chains at this temperature (25 degrees C). The DPPC (54%)/PS (36%)/CPZ (10%) bilayer at a sample temperature of 37 degrees C (T(C)=31.2 degrees C), shows CPZ interdigitation among the phospholipids as deduced from the finding that approximately 30% of the phospholipid acyl chains carbon resonances shift low-field by 5-15 ppm.     

Biophysical studies on chitosan-coated liposomes

Liposomes have been used as delivery vehicles for stabilizing drugs, overcoming barriers to cellular and tissue uptake, and for directing their contents toward specific sites in vivo. Chitosan is a biological macromolecule derived from crustacean shells and has several emerging applications in drug development, obesity control, and tissue engineering. In the present work, the interaction between chitosan and dipalmitoyl phosphatidylcholine (DPPC) liposomes was studied by transmission electron microscopy (TEM), zeta potential, solubilization using the nonionic detergent octylglucoside (OG), as well as Fourier transform infrared (FTIR) spectroscopy and viscosity measurements. The coating of DPPC liposomes by a chitosan layer was confirmed by electron microscope images and the zeta potential of liposomes. Coating of liposome by chitosan resulted in an increase in liposomal size by addition of a layer of 92 ± 27.1 nm. The liposomal zeta potential became increasingly positive as chitosan concentration increased from 0.1 to 0.3% w/v, then it held at a relatively constant value. The amount of detergent needed to completely solubilize the liposomal membrane was increased after coating of liposomes with chitosan, indicating an increased membrane resistance to the detergent and hence a change in the natural membrane permeation properties. In the analysis of FTIR spectra of DPPC, the symmetric and antisymmetric CH₂ (at 2,800–3,000 cm⁻¹) bands and the C=O (at 1,740 cm⁻¹) stretching band were investigated in the absence and presence of the chitosan. It was concluded that appropriate combining of the liposomal and chitosan characteristics might be utilized for the improvement of the therapeutic efficacy of liposomes as a drug delivery system.     

Dye permeability at phase transitions in single and binary component phospholipid bilayers

By encapsulating a pH-sensitive dye, phenol red, in multilamellar liposomes of DMPC, DPPC and DMPC/DPPC mixtures, the permeability of these phospholipid bilayers to dye as a function of temperature has been studied. For both DMPC and DPPC liposomes, dye release begins well below the main gel-to-liquid-crystalline phase transition (24°C and 42°C, respectively) at temperatures corresponding to the onset of the pretransition (about 14°C and 36°C, respectively) with DPPC liposomes exhibiting a permeability anomaly at the main phase transition (42°C). The perturbation occurring in the bilayer structure that allows the release of encapsulated phenol red (approx. 5 Å diameter) is not sufficient to permit the release of encapsulated haemoglobin (approx. 20 Å diameter, negatively charged). In liposomes composed of a range of DMPC/DPPC mixtures, dye release commences at the onset of the pretransition range (determined by optical absorbance measurements) and increases with increasing temperature until the first appearance of liquid crystalline phase after which no further dye release occurs. Interestingly, the dye retaining properties of DMPC and DPPC liposomes well below their respective pretransition temperature regions are very different: DMPC liposomes release much encapsulated dye at incubation temperatures of 5°C whilst DPPC liposomes do not.     

The influence of selected steroid hormones on the physicochemical behaviour of DPPC liposomes

The physicochemical properties of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) liposomes used for topical application are pharmaceutically important. Therefore the aim of our study was to establish rapid and efficient methods for the exact characterisation of the physicochemical properties of extruded DPPC liposomes containing low concentration (0.5%, w/w) of different, therapeutically interesting steroid hormones, named 17-beta-estradiol, cpa (cyproterone acetate) and finasteride. In a first step it could be shown that all drugs influenced the liposome size and changed the zeta potential compared to the placebo formulations. Our further analytical strategy was to use micro-calorimetry and ATR-FTIR (Fourier transformed infrared spectroscopy), two powerful and non-destructive methods to confirm the drug incorporation into the liposomes by proving interactions between the phospholipids and the steroids. Thereby it was even possible to localize the location of interaction. The characteristic phase transition temperatures of the phospholipid were decreased by the hormones which was detected by micro-DSC (differential scanning calorimetry). The results of the ATR-FTIR measurements indicated shifts of the specific lipid peaks, the C=O stretching bands and PO(2)(-) antisymmetric double stretching band, in the gel and liquid crystalline phase. A polar as well as a non-polar interaction could be proven. It could be shown that the investigated steroid hormones changed the physical properties of the phospholipid bilayers.     

Chitosan matrix with three dimensionally ordered macroporous structure for nimodipine release

Three dimensionally ordered macroporous (3DOM) chitosan (3D-CS) matrix with interconnected pores in the nanometer range was developed as a drug carrier for the first time. 3D-CS was prepared using a template-assisted assembly and characterized by SEM, TGA, N(2) adsorption and FT-IR. As a model drug, nimodipine (NMDP) was incorporated into the pores of 3D-CS matrix. The solid state properties of NMDP-loaded samples were characterized by SEM, XRD, DSC and FT-IR. Dissolution studies showed that release behavior of the drug was markedly affected by the particle size of the matrix. With a relatively small matrix particle size, formulations of NMDP-3D-CS-0.5 and NMDP-3D-CS-1 exhibited rapid release patterns. However, on increasing the amount of carrier, release rate of the drug decreased. The pH-dependent slow-release characteristic of 3D-CS matrix delivery system was demonstrated by investigating the release behavior of NMDP at different pH values.     

Drug-induced zeta potential changes in liposomes studied by laser Doppler spectroscopy

The technique of laser Doppler spectroscopy is used to measure the electrophoretic mobility of liposomes under the influence of one beta-blocking agent and three local anesthetics. All four drugs decrease the mobility (i.e., the zeta potential) of negatively charged phospholipids (soybean lipids, phosphatidylserine and cardiolipin). The mobility of electrostatically neutral pure phosphatidylcholine (zero mobility under control conditions at pH 7 and 4) is increased linearly with the logarithm of drug concentration, indicating binding and incorporation of positively charged drug molecules. The sequence of strength of activity, measured by zeta-potential changes, corresponds to that found in biological tissues: propranolol greater than tetracaine greater than lidocaine greater than procaine. For purely negatively charged lipids (phosphatidylserine, cardiolipin) the activity of the drug is higher at acidic pH, (pH 4), while for electrostatically neutral (phosphatidylcholine) or partly neutral (soybean) lipid liposomes drug activity is about the same at pH 9, 7 and 4. A Hill plot of the data reveals noncooperative drug binding. From the line width of the scattering power spectrum the mean particle radius and the average interparticle distance in the samples are determined.     

Modification of glyceraldehyde 3-phosphate dehydrogenase activity by adsorption on phospholipid vesicles.

1. The adsorption of [14C]carboxymethylated glyceraldehyde 3-phosphate dehydrogenase to negatively charged liposomes of phsphatidic acid/phosphatidylcholine (3:7, w/w) was investigated. The apparent association constant at I/2 = 60, pH 7.6, was 0.4 X 10(6)M-1. Adsorption decreased as ionic strength and pH were increased. 2. In the presence of negatively charged liposomes, the Km value for glyceraldehyde 3-phosphate of glyceraldehyde 3-phosphate dehydrogenase was increased and Vmax. decreased. In the presence of positively charged liposomes, the Km value for glyceraldehyde 3-phosphate decreased and there was no significant change in Vmax. Addition of Triton X-100 abolished the effect of both positively and negatively charged liposomes on the kinetic properties of the enzyme.     

Preparation and characterization of gas-filled liposomes: can they improve oil recovery?

Although well known for delivering various pharmaceutical agents, liposomes can be prepared to entrap gas rather than aqueous media and have the potential to be used as pressure probes in magnetic resonance imaging (MRI). Using these gas-filled liposomes (GFL) as tracers, MRI imaging of pressure regions of a fluid flowing through a porous medium could be established. This knowledge can be exploited to enhance recovery of oil from the porous rock regions within oil fields. In the preliminary studies, we have optimized the lipid composition of GFL prepared using a simple homogenization technique and investigated key physico-chemical characteristics (size and the physical stability) and their efficacy as pressure probes. In contrast to the liposomes possessing an aqueous core which are prepared at temperatures above their phase transition temperature (T(c)), homogenization of the phospholipids such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocoline (DSPC) in aqueous medium below their T(c) was found to be crucial in formation of stable GFL. DSPC based preparations yielded a GFL volume of more than five times compared to their DPPC counter part. Although the initial vesicle sizes of both DSPC and DPPC based GFL were about 10 microm, after 7 days storage at 25 degrees C, the vesicle sizes of both formulations significantly (p < 0.05) increased to 28.3 +/- 0.3 mum and 12.3 +/- 1.0 microm, respectively. When the DPPC preparation was supplemented with cholesterol at a 1:0.5 or 1:1 molar ratio, significantly (p < 0.05) larger vesicles were formed (12-13 microm), however, compared to DPPC only vesicles, both cholesterol supplemented formulations displayed enhanced stability on storage indicating a stabilizing effect of cholesterol on these gas-filled vesicles. In order to induce surface charge on the GFL, DPPC and cholesterol (1: 0.5 molar ratio) liposomes were supplemented with a cationic surfactant, stearylamine, at a molar ratio of 0.25 or 0.125. Interestingly, the zeta potential values remained around neutrality at both stearylamine ratios suggesting the cationic surfactant was not incorporated within the bilayers of the GFL. Microscopic analysis of GFL confirmed the presence of spherical structures with a size distribution between 1-8 microm. This study has identified that DSPC based GFL in aqueous medium dispersed in 2% w/v methyl cellulose although yielded higher vesicle sizes over time were most stable under high pressures exerted in MRI.     

Physical biochemistry of a liposomal amphotericin B mixture used for patient treatment

There seems little doubt now that intravenous liposomal amphotericin B can be a useful treatment modality for the management of immunocompromised patients with suspected or proven disseminated fungal infections. Interestingly, the very significant reduction in toxicity reported when amphotericin B is part of a bilayer membrane is closely tied to the physical characteristics of the liposomes involved, although these are poorly understood at the molecular level. We record here an examination by spectroscopy and freeze-etch electron microscopy of unsonicated amphotericin B multilamellar vesicles prepared along the lines that we and others have followed for samples used in clinical trials and preclinical in vivo or in vitro studies. Our study has focussed on liposomes of 7:3 dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) bearing 0-25 mol% amphotericin B, since this lipid mixture has been the choice for the first clinical trials. Phase transition behaviour of these liposomes was examined by electron paramagnetic resonance (EPR) spectroscopy of a nitroxide spin label partitioning into the bilayers. The same experiments were then performed on similarly prepared liposomes of the disaturated species, dipalmitoylphosphatidylcholine (DPPC), and the diunsaturated species, dielaidoylphosphatidylcholine (DEPC). Partial phase diagrams were constructed for each of the lipid/drug mixtures. Melting curves and derived phase diagrams showed evidence that amphotericin B is relatively immiscible with the solid phase of bilayer membranes. The phase diagram for DEPC/amphotericin B was very similar to that of DPPC/amphotericin B, and both exhibited less extensive temperature ranges of phase separation than did the 7:3 DMPC/DMPG mixture with amphotericin B. Between 25 and 37 degrees C the measured fluidity of the 7:3 DMPC/DMPG liposomes was similar to that of the (unsaturated fatty acid) DEPC liposomes, and considerably higher than that seen for (saturated fatty acid) DPPC liposomes. Preparations of 7:3 DMPC/DMPG, DPPC, and DEPC containing 0-25 mol% amphotericin B were examined by freeze-etch electron microscopy at 35 and 22 degrees C (to cover the temperature range of the mammalian body core and periphery). The same liposome features were present in all three liposome types studied. The appearance of individual liposomes at x 100,000 magnification reflected their molecular characteristics, which were found to be significantly heterogeneous within each batch. The lipid/drug structures were bilayer in nature, although liposomes showing considerable disruption were common, particularly at the highest drug concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development and modeling of arsenic-trioxide–loaded thermosensitive liposomes for anticancer drug delivery

In this article, a novel delivery system for the anticancer drug, arsenic trioxide (ATO), is characterized. The release of ATO from DPPC liposomes with MPPC lysolipid incorporated into the bilayer was measured. Upon heating the liposomes to 37°C, there was a 15–25% release over 24 hours. The ATO release from the DPPC and DPPC:MPPC (5%) systems leveled off after 10 hours at 37°C, whereas the DPPC:MPPC (10%) liposomes continue to release ATO over the 24-hour time span. Upon heating the liposomes rapidly to 42°C, the release rate was substantially increased. The systems containing lysolipids exhibited a very rapid release of a significant amount of arsenic in the first hour. In the first hour, the DPPC:MPPC (5%) liposomes released 40% of the arsenic and the DPPC:MPPC (10%) liposomes released 55% of the arsenic. Arsenic release from pure DPPC liposomes was comparable at 37 and 42°C, indicating that the presence of a lysolipid is necessary for a significant enhancement of the release rate. A coarse-grained molecular dynamics (CGMD) model was used to investigate the enhanced permeability of lysolipid-incorporated liposomes and lipid bilayers. The CG liposomes did not form a gel phase when cooled due to the high curvature; however, permeability was still significantly lower below the liquid-to-gel phase-transition temperature. Simulations of flat DPPC:MPPC bilayers revealed that a peak in the permeability did coincide with the phase transition from the gel to LC state when the lysolipid, MPPC, was present. No pores were observed in the simulations, so it is unlikely this was the permeability-enhancing mechanism.     

Effects of phospholipid hydrolysis on the aggregate structure in DPPC/DSPE-PEG2000 liposome preparations after gel to liquid crystalline phase transition

Upon storage of phospholipid liposome samples, lysolipids, fatty acids, and glycerol-3-phosphatidylcholine are generated as a result of acid- or base-catalyzed hydrolysis. Accumulation of hydrolysis products in the liposome membrane can induce fusion, leakage, and structural transformations of the liposomes, which may be detrimental or beneficial to their performance depending on their applications as, e.g., drug delivery devices. We investigated in the present study the influence of phospholipid hydrolysis on the aggregate morphology of DPPC/DSPE-PEG2000 liposomes after transition of the phospholipid membrane from the gel phase to liquid crystalline phase using high performance liquid chromatography (HPLC) in combination with static light scattering, dynamic light scattering, and cryo-transmission electron microscopy (cryo-TEM). The rates of DPPC hydrolysis in DPPC/DSPE-PEG2000 liposomes were investigated at a pH of 2, 4, or 6.5 and temperatures of 22 degrees C or 4 degrees C. Results indicate that following phase transition, severe structural reorganizations occurred in liposome samples that were partially hydrolyzed in the gel phase. The most prominent effect was an increasing tendency of liposomes to disintegrate into membrane discs in accordance with an increasing degree of phospholipid hydrolysis. Complete disintegration occurred when DPPC concentrations had decreased by, in some cases, as little as 3.6%. After extensive phospholipid hydrolysis, liposomes and discs fused to form large bilayer sheets as well as other more complex bilayer structures apparently due to a decreased ratio of lysolipid to palmitic acid levels in the liposome membrane.     

Improved methods for the delivery of liposome-sequestered RNA into eucaryotic cells

RNA sequestered by negatively charged liposomes becomes cell-associated following interaction between eucaryotic cells and the liposomes. This paper provides evidence that cell-associated RNA is internalized by the cells. In fact, (a) when Escherichia coli and mammalian RNA are entrapped within the same liposome population and delivered into cultured cells, one can observe degradation of the procaryotic but not the eucaryotic RNA. Such an event cannot happen extracellularly. (b) Scanning electron microscopy reveals no more than 10 liposomes adhering to each cell upon liposome-cell interaction under conditions in which the RNA entrapped by 140 liposomes becomes associated with each cell. The ability of liposomes prepared by (a) the cochleate process, (b) the reverse-phase evaporation technique, and (c) the ether infusion technique, to sequester and deliver RNA into cells was investigated. Reverse-phase evaporated liposomes were most efficient in sequestering RNA (20–40%), however, all types of liposomes delivered RNA with comparable efficiency. The rate of liposome-mediated RNA delivery into mammalian cells could be substantially improved when: (a) liposome-cell interaction was carried out at pH 6.5 (twofold increase over pH 7.5), (b) a basic protein (methylated albumin) was present (two- to threefold increase), (c) liposome-cell cultures were treated with polyethylene glycol 6000 (four- to eight-fold increase), and (d) DEAE-dextran was added during interaction of liposomes with cell monolayers (four- to eight-fold increase).     

Effects of phospholipid hydrolysis on the aggregate structure in DPPC/DSPE-PEG2000 liposome preparations after gel to liquid crystalline phase transition

Upon storage of phospholipid liposome samples, lysolipids, fatty acids, and glycerol-3-phosphatidylcholine are generated as a result of acid- or base-catalyzed hydrolysis. Accumulation of hydrolysis products in the liposome membrane can induce fusion, leakage, and structural transformations of the liposomes, which may be detrimental or beneficial to their performance depending on their applications as, e.g., drug delivery devices. We investigated in the present study the influence of phospholipid hydrolysis on the aggregate morphology of DPPC/DSPE-PEG₂₀₀₀ liposomes after transition of the phospholipid membrane from the gel phase to liquid crystalline phase using high performance liquid chromatography (HPLC) in combination with static light scattering, dynamic light scattering, and cryo-transmission electron microscopy (cryo-TEM). The rates of DPPC hydrolysis in DPPC/DSPE-PEG₂₀₀₀ liposomes were investigated at a pH of 2, 4, or 6.5 and temperatures of 22 °C or 4 °C. Results indicate that following phase transition, severe structural reorganizations occurred in liposome samples that were partially hydrolyzed in the gel phase. The most prominent effect was an increasing tendency of liposomes to disintegrate into membrane discs in accordance with an increasing degree of phospholipid hydrolysis. Complete disintegration occurred when DPPC concentrations had decreased by, in some cases, as little as 3.6%. After extensive phospholipid hydrolysis, liposomes and discs fused to form large bilayer sheets as well as other more complex bilayer structures apparently due to a decreased ratio of lysolipid to palmitic acid levels in the liposome membrane.     

Effect of the fungicides tributyltin acetate and tributyltin chloride on multilamellar liposomes: fluorescence studies.

The influence of tri-n-butyltin acetate (TBTA) and tri-n-butyltin chloride (TBTC) on the physico-chemical state of charged and neutral phospholipids was investigated using multilamellar liposomes. The thermal dependence of steady state fluorescence polarization of DPH and its charged derivative TMA-DPH was recorded. The two fungicides lowered DPPC phase transition temperature and broadened the temperature range of the transition in different ways. The effects were concentration-dependent. The results show that TBTC interacts more effectively with DPPC model membranes rather than TBTA. Moreover, TBTC broadens and shifts the main phase transition (Tm) more effectively in DPPC rather than in DMPC liposomes. Below Tm, TBTC decreases fluorescence polarization (P) in all phospholipids used. Above Tm P is almost constant in phospholipids with saturated acyl chains, except for DMPG. In fact, an increase of P is detectable in this lipid as in PLs with unsaturated acyl chains. It is suggested that the effects of TBT on liposomal membranes are dependent on the anion moiety and phospholipids characteristics.