Biotin and glucose dual-targeting,ligand-modified liposomes promote breast tumor-specific drug delivery

Breast cancer is the second leading cause of cancer-related deaths in women. Ligand-modified liposomes are used for breast tumor-specific drug delivery to improve the efficacy and reduce the side effects of chemotherapy; however, only a few liposomes with high targeting efficiency have been developed because the mono-targeting, ligand-modified liposomes are generally unable to deliver an adequate therapeutic dose. In this study, we designed biotin-glucose branched ligand-modified, dual-targeting liposomes (Bio-Glu-Lip) and evaluated their potential as a targeted chemotherapy delivery system in vitro and in vivo. When compared with the non-targeting liposome (Lip), Bio-Lip, and Glu-Lip, Bio-Glu-Lip had the highest cell uptake in 4T1 cells (3.00-fold, 1.60-fold, and 1.95-fold higher, respectively) and in MCF-7 cells (2.63-fold, 1.63-fold, and 1.85-fold higher, respectively). The subsequent cytotoxicity and in vivo assays further supported the dual-targeting liposome is a promising drug delivery carrier for the treatment of breast cancer.     

Tissue plasminogen activator catalyzed Lys-plasminogen activation on heparin-inserted phospholipid liposomes

We prepared heparin-inserted phospholipid liposomes as a functional model of heparan sulfate present on the vascular surface and examined tissue plasminogen activator (t-PA) catalyzed plasminogen activation on the liposome surface. Kinetic analyses showed a marked increase in the affinity of t-PA for Lys-plasminogen in the presence of heparin-inserted phosphatidylcholine (PC) liposomes. The catalytic efficiency (kcat/Km) of t-PA for the plasminogen activation on the surface of heparin-inserted PC liposomes was 5.4 times that on the surface of heparin-free PC liposomes. This stimulatory action of immobilized heparin was apparently affected by changing the phospholipid component of liposomes. Phosphatidylethanolamine or stearylamine, having a positively charged group, reduced the catalytic efficiency of t-PA by raising its Km value (10-fold), whereas negatively charged phospholipids, phosphatidylserine and phosphatidylinositol, did not affect the efficiency. t-PA and generated plasmin bound to the liposome surface heparin were protected from inhibition by plasminogen activator inhibitor type 1 and alpha 2-plasmin inhibitor, respectively. t-PA-induced clot lysis of euglobulin or whole plasma, which contained native (Glu-) plasminogen and the above inhibitors, was also accelerated by addition of heparin-inserted PC liposomes. These results suggest that the vascular surface heparin-like molecules may play an important role in modulating fibrinolytic events. The principles of conjugation of t-PA with a biologically active liposome will be applied to the construction of better thrombolytic agents.     

Receptor-mediated uptake of asialoganglioside liposomes: sub-cellular distribution of the liposomal marker in isolated liver cell types

The use of asialo GM1-containing small unilamellar liposome preparations in vivo caused a 2.8-fold increase in the uptake by the liver as compared with the control (neutral) preparations (without asialo GM1). The uptake of negatively charged dicetylphosphate and dipalmitoyl phosphatidic acid-containing small unilamellar liposomes was found to be 1.6-and 1.8-fold respectively higher than that of the neutral preparations. In studies with isolated liver cell types, inhibition of the galactosylated liposome uptake by asialofetuin indicated a possible involvement of hepatic galactose receptors in the recognition of asialo GM1 liposomes by the hepatic parenchymal cells, which in turn were found to be mainly responsible for the enhanced incorporation of these liposomes in the liver. Sub-cellular distribution studies with isolated liver cell types indicated lysosomal localization of the liposomes both in parenchymal and nonparenchymal cells, and it has been proposed that the asialo GM1 liposomes are cointernalized with asialofetuin through a common lysosomal route of ligand internalization.     

Gene transfection efficiency of tracheal epithelial cells by DC-chol-DOPE/DNA complexes.

We evaluated the transfection efficiency of five different cationic liposome/plasmid DNA complexes, during the in vitro gene transfer into human epithelial tracheal cell lines. A dramatic correlation between the transfection efficiency and the charge ratio (positive charge of liposome to negative charge of DNA) has been found. DC-Chol-DOPE was found to be the most effective liposome formulation. Therefore, a morphological and structural analysis of DC-Chol-DOPE liposomes and DC-Chol-DOPE/DNA complexes, has been performed by transmission electron microscopy (TEM) and by confocal laser scanning microscopy (CLSM), respectively. The process of interaction between DC-Chol-DOPE/DNA complexes and human epithelial tracheal cells has been studied by CLSM. These results raise some issues for in vivo gene therapy.     

Ethanol injection method for hydrophilic and lipophilic drug-loaded liposome preparation

In this article, a hydrophobic (beclomethasone dipropionate; BDP) and a hydrophilic (cytarabine; Ara-C) drugs have been encapsulated in liposomes in order to be administered via the pulmonary route. For this aim, a liposome preparation method, which is easy to scale up, the ethanol injection method, has been selected. The effects of critical process and formulation parameters have been investigated. The drug-loaded liposomes were prepared and characterized in terms of size, zeta potential, encapsulation efficiency, release study, cell uptake, and aerodynamic behavior. Small multilamellar vesicles, with sizes ranging from about 80 to 170?nm, were successfully obtained. Results indicated a significant influence of phospholipid and cholesterol amounts on liposome size and encapsulation efficiency. The higher encapsulation efficiencies were about 100% for the hydrophobic drug (BDP) and about 16% for the hydrophilic one (Ara-C). The in vitro release study showed a prolonged release profile for BDP, in contrast with Ara-C, which was released more rapidly. The cell-uptake test revealed that fluorescent liposomes have been well internalized into the cytoplasm of SW-1573 human lung carcinoma cells, confirming the possibility to use liposomes for lung cell targeting. Nebulized Ara-C and BDP liposomes presented aerodynamic diameters compatible with deep lung deposition. In conclusion, the elaborated liposomes seem to be promising carriers for both Ara-C and BDP pulmonary delivery.     

Tissue distribution of EDTA encapsulated within liposomes containing glycolipids or brain phospholipids.

Multilameller liposomes were prepared with various asialoglycolipids, gangliosides, sialic acid, or brain phospholipids in the liposome membrane and with ethylenediaminetetraacetic acid (EDTA) encapsulated in the aqueous compartments. The liposomes containing glycolipids or sialic acid were prepared from a mixture of phosphatidylcholine, cholesterol, and one of the following test substances: galactocerebroside, glucocerebroside, galactocerebroside sulfate, mixed gangliosides, monosialoganglioside GM1, monosialoganglioside GM2, monosialoganglioside GM3, disialoganglioside GD1a, or sialic acid. The liposomes containing brain phospholipids were mixtures of either sphingomyelin and cholesterol or a brain total phospholipid extract and cholesterol. Distributions of 14C-labeled EDTA were determined in mouse tissues from 15 min to 6 h or 12 h after a single injection of liposome preparation. Liver uptake up encapsulated EDTA was lowest from all liposome preparations containing sialic acid or sialogangliosides, regardless of the amount of sialic acid moiety present or the identity of the particular ganglioside; highest uptake of encapsulated EDTA by liver was from liposomes containing galactocerebroside or brain phospholipids. Lungs and brain took up the largest amounts of EDTA from liposomes containing sphingomyelin and lesser amounts from liposomes containing GD1a. Use of mouse brain phospholipid extract to prepare liposomes did not increase uptake of encapsulated EDTA by the brain. EDTA in liposomes containing monosialogangliosides, brain phospholipids, galactocerebroside, or sialic acid was taken up well by spleen and marrow. Highest thymus uptake of encapsulated EDTA was from liposomes containing GD1a. These results demonstrate that inclusion of sialogangliosides in liposome membranes decreases uptake of liposomes by liver, thus making direction of encapsulated drugs to other organs more feasible. Liposomes containing glycolipids also have potential uses as probes of cell surface receptors.     

Essential oils encapsulated in liposomes: a review

Abstract

In the recent years there has been an increased interest toward the biological activities of essential oils. However, essential oils are unstable and susceptible to degradation in the presence of oxygen, light and temperature. So, attempts have been made to preserve them through encapsulation in various colloidal systems such as microcapsules, microspheres, nanoemulsions and liposomes. This review focuses specifically on encapsulation of essential oils into liposomes. First, we present the techniques used to prepare liposomes encapsulating essential oils. The effects of essential oils and other factors on liposome characteristics such as size, encapsulation efficiency and thermal behavior of lipid bilayers are then discussed. The composition of lipid vesicles membrane, especially the type of phospholipids, cholesterol content, the molar ratio of essential oils to lipids, the preparation method and the kind of essential oil may affect the liposome size and the encapsulation efficiency. Several essential oils can decrease the size of liposomes, homogenize the liposomal dispersions, increase the fluidity and reduce the oxidation of the lipid bilayer. Moreover, liposomes can protect the fluidity of essential oils and are stable at 4–5?°C for 6 months at least. The applications of liposomes incorporating essential oils are also summarized in this review. Liposomes encapsulating essential oils are promising agents that can be used to increase the anti-microbial activity of the essential oils, to study the effect of essential oils on cell membranes, and to provide alternative therapeutic agents to treat several diseases.     

Liposome-mediated delivery of deoxyribonucleic acid to cells: enhanced efficiency of delivery related to lipid composition and incubation conditions

Delivery of liposome-encapsulated simian virus 40 (SV40) DNA to African green monkey Related to been used as a probe to study liposome--cell interactions and to determine conditions which favor the intracellular delivery of liposome contents to cells. The efficiency of DNA delivery by various liposome preparations (monitored by infectivity assays) was found to be dependent both on the magnitude of vesicle binding to cells and on the resistance of liposomes to cell-induced leakage of contents. Acidic phospholipids were much more effective in both binding and delivery, and phosphatidylserine (PS) was the best in both aspects. The inclusion of 50 mol % cholesterol in liposomes reduces the cell-induced leakage of vesicle contents (2--5-fold) and substantially enhances the delivery of DNA to cells (2--10-fold). Following incubation of cells with negatively charged liposomes containing SV40 DNA, infectivity can be enhanced greatly by brief exposure of the cells to glycerol solutions. In contrast, only slight enhancement by glycerol was observed for SV40 DNA encapsulated in neutral or positively charged liposomes. The results of competition experiments between empty phosphatidylcholine liposomes and DNA-containing PS liposomes also suggest possible differences in the interaction of neutral and negatively charged liposome preparations with cells. Morphological studies indicate that the glycerol treatment stimulates membrane ruffling and vacuolization and suggest that the enhanced uptake of liposomes occurs by an endocytosis-like process. Results obtained with metabolic inhibitors are also consistent with the interpretation that the enhancement of liposome delivery in glycerol-treated cells occurs via an energy-dependent endocytotic pathway. Pretreatment of cells with chloroquine, a drug which alters lysosomal activity, further enhanced infectivity in glycerol-treated cells (4-fold). This observation suggests the involvement of a lysosomal processing step at some point in the expression of liposome-encapsulated DNA and, more importantly, illustrates the possibility of altering cellular mechanism to engineer more efficient delivery by liposomes. Under optimal conditions determined in this study, the efficiency of liposome-mediated SV40 DNA delivery was increased more than 1000-fold over that obtained by simply incubating cells with liposomes. It is also demonstrated that these conditions enhance delivery of other molecules, besides DNA, which are encapsulated in liposomes.     

Protein-liposome conjugates using cysteine-lipids and native chemical ligation

Liposomes have become popular drug delivery vehicles and have more recently also been applied as contrast agents for molecular imaging. Most current methods for functionalization of liposomes with targeting proteins rely on reactions of amine or thiol groups at the protein exterior, which generally result in nonspecific conjugation at multiple sites on the protein. In this study, we present native chemical ligation (NCL) as a general method to covalently couple recombinant proteins in a highly specific and chemoselective way to liposomes containing cysteine-functionalized phospholipids. A cysteine-functionalized phospholipid (Cys-PEG-DSPE) was prepared and shown to readily react with the MESNA thioester of EYFP, which was used as a model protein. Characterization of the EYFP-liposomes using fluorescence spectroscopy showed full retention of the fluorescent properties of conjugated EYFP and provides a lower limit of 120 proteins per liposome. The general applicability of NCL was further tested using CNA35, a collagen-binding protein recently applied in fluorescent imaging of collagen. NCL of CNA35 thioester yielded liposomes containing approximately 100 copies of CNA35 per liposome. The CNA35-liposomes were shown to be fully functional and bind collagen with a 150-fold higher affinity compared to CNA35. Our results show that NCL is an attractive addition to existing conjugation methods that allows direct, covalent, and highly specific coupling of recombinant proteins to liposomes and other lipid-based assemblies.     

Highly efficient DNA delivery mediated by pH-sensitive immunoliposomes

We have previously shown that pH-sensitive immunoliposomes can mediate a target-specific delivery of plasmid DNA to tumor cells grown in a mouse model [Wang, C.-Y., & Huang, L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7851-7855]. The efficiency of delivery in terms of the target cell transformation frequency has now been characterized for both short- and long-term gene expression in a tissue culture system. Herpes simplex virus thymidine kinase (TK) gene was used as a reporter gene. It was placed under the control of the promoter for the rat phosphoenolpyruvate carboxykinase gene, which contains a cAMP regulatory element. Therefore, the expression of the exogenous gene in the target cell, mouse Ltk- cells, can be regulated by cAMP drugs. The plasmid DNA was encapsulated in liposomes using a detergent dialysis method. The efficiency of gene delivery was optimized with respect to the time course and dose of liposome-associated DNA. The existence of antibody of the liposomes was essential for the maximal level of DNA delivery. Delivery was also dependent on the lipid composition of the liposome. The pH-sensitive lipid composition gave 8-fold higher efficiency than the corresponding pH-insensitive composition. The transformation efficiency of the target cell also depended on the regulation of gene expression; cells incubated with dibutyryl-cAMP and theophylline showed a much higher level of transformation frequency than cells incubated without the drugs. When all liposome and incubation parameters are optimized, the Ltk- cells showed a 47% efficiency for the short-term transformation, and 2% for the long-term transformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of lipid composition on the structure and theoretical phase diagrams of DC-Chol/DOPE-DNA lipoplexes

Lipoplexes constituted by calf-thymus DNA (CT-DNA) and mixed cationic liposomes consisting of varying proportions of the cationic lipid 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol) and the zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphoetanolamine (DOPE) have been analyzed by means of electrophoretic mobility, SAXS, and fluorescence anisotropy experiments, as well as by theoretically calculated phase diagrams. Both experimental and theoretical studies have been run at several liposome and lipoplex compositions, defined in terms of cationic lipid molar fraction, α, and either the mass or charge ratios of the lipoplex, respectively. The experimental electrochemical results indicate that DC-Chol/DOPE liposomes, with a mean hydrodynamic diameter of around (120 ± 10) nm, compact and condense DNA fragments at their cationic surfaces by means of a strong entropically driven electrostatic interaction. Furthermore, the positive charges of cationic liposomes are compensated by the negative charges of DNA phosphate groups at the isoneutrality L/D ratio, (L/D)(?), which decreases with the cationic lipid content of the mixed liposome, for a given DNA concentration. This inversion of sign process has been also studied by means of the phase diagrams calculated with the theoretical model, which confirms all the experimental results. SAXS diffractograms, run at several lipoplex compositions, reveal that, irrespectively of the lipoplex charge ratio, DC-Chol/DOPE-DNA lipoplexes show a lamellar structure, L(α), when the cationic lipid content on the mixed liposomes α ≥ 0.4, while for a lower content (α = 0.2) the lipoplexes show an inverted hexagonal structure, H(II), usually related with improved cell transfection efficiency. A similar conclusion is reached from fluorescence anisotropy results, which indicate that the fluidity on liposome and lipoplexes membrane, also related with better transfection results, increases as long as the cationic lipid content decreases.     

Tissue distribution of EDTA encapsulated within liposomes containing glycolipids or brain phospholipids

Multilamellar liposomes were prepared with various asialoglycolipids, gangliosides, sialic acid, or brain phospholipids in the liposome membrane and with ethylenediaminetetraacetic acid (EDTA) encapsulated in the aqueous compartments. The liposomes containing glycolipids or sialic acid were prepared from a mixture of phosphatidylcholine, cholesterol, and one of the following test substances: galactocerebroside, glucocerebroside, galactocerebroside sulfate, mixed gangliosides, monosialoganglioside G_M1, monosialoganglioside G_M2, monosialoganglioside G_M3, disialoganglioside G_D1a, or sialic acid. The liposomes containing brain phospholipids were mixtures of either sphingomyelin and cholesterol or a brain total phospholipid extract and cholesterol. Distribution of ¹⁴C-labeled EDTA were determined in mouse tissues from 15 min to 6 h or 12 h after a single injection of liposome prepartion. Liver uptake of encapsulated EDTA was lowest from all liposome preparations containing sialic acid or sialogangliosides regardless of the amount of sialic acid moiety present or the identity of the particular ganglioside; highest uptake of encapsulated EDTA by liver was from the liposomes containing galactocerebroside or brain phospholipids. Lungs and brain took up the largest amounts of EDTA from liposomes containing sphingomyelin and lesser amounts from liposomes containing G_D1a. Use of mouse brain phospholipid extract to prepare liposomes did not increase uptake of encapsulated EDTA by the brain. EDTA in liposomes containing monosialogangliosides, brain phospholipids, galactocerebroside, or sialic acid was taken up well by spleen and marrow. Highest thymus uptake of encapsulated EDTA was from liposomes containing G_D1a. These results demonstrate that inclusion of sialogangliosides in liposome membranes decreases uptake of liposomes by liver, thus making direction of encapsulated drugs to other organs more feasible. Liposomes containing glycolipids also have potential uses as probes of cell surface receptors.     

Syntheses and properties of circular dichroism active phospholipids

A group of circular dichroism (CD) active phospholipids has been synthesised, in which one or both acyl chains has been replaced with a cinnamoyl or azobenzene chromophore-containing acid. Studies on the structure, CD activity and thermodynamic property of liposome membranes composed of CD active phospholipids were carried out. CD active liposomes were found to be stable, normal liposomes of approximately 550 A diameter based on the electron micrograph and dynamic light scattering, and to have thermodynamic property similar to the conventional phospholipid membranes without serious perturbation by aromatic bulk groups based on DSC. Liposomes composed of phospholipid having two trans-azobenzene chromophores showed an extremely large CD enhancement even well above Tc. This CD enhancement was drastically changed by the presence of cis-azobenzene chromophore and cis-cis isomer content after irradiation was higher than the theoretical value, suggesting the importance of interchromophore interaction in the liposome membranes.     

Effects of phospholipid composition on MinD-membrane interactions in vitro and in vivo

The peripheral membrane ATPase MinD is a component of the Min system responsible for correct placement of the division site in Escherichia coli cells. By rapidly migrating from one cell pole to the other, MinD helps to block unwanted septation events at the poles. MinD is an amphitropic protein that is localized to the membrane in its ATP-bound form. A C-terminal domain essential for membrane localization is predicted to be an amphipathic alpha-helix with hydrophobic residues interacting with lipid acyl chains and cationic residues on the opposite face of the helix interacting with the head groups of anionic phospholipids (Szeto, T. H., Rowland, S. L., Rothfield, L. I., and King, G. F. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 15693-15698). To investigate whether E. coli MinD displays a preference for anionic phospholipids, we first examined the localization dynamics of a green fluorescent protein-tagged derivative of MinD expressed in a mutant of E. coli that lacks phosphatidylethanolamine. In these cells, which contain only anionic phospholipids (phosphatidylglycerol and cardiolipin), green fluorescent protein-MinD assembled into dynamic focal clusters instead of the broad zones typical of cells with normal phospholipid content. In experiments with liposomes composed of only zwitterionic, only anionic, or a mixture of anionic and zwitterionic phospholipids, purified MinD bound to these liposomes in the presence of ATP with positive cooperativity with respect to the protein concentration and exhibited Hill coefficients of about 2. Oligomerization of MinD on the liposome surface also was detected by fluorescence resonance energy transfer between MinD molecules labeled with different fluorescent probes. The affinity of MinD-ATP for anionic liposomes as well as liposomes composed of both anionic and zwitterionic phospholipids increased 9- and 2-fold, respectively, relative to zwitterionic liposomes. The degree of acyl chain unsaturation contributed positively to binding strength. These results suggest that MinD has a preference for anionic phospholipids and that MinD oscillation behavior, and therefore cell division site selection, may be regulated by membrane phospholipid composition.     

The effect of aging on the physical stability of liposome dispersions.

In this study, the effect of aging, in terms of hydrolytic decomposition of the bilayer forming (phospho)lipids, on the physical stability of aqueous liposome dispersion was investigated in partially hydrogenated egg phosphatidylcholine (PHEPC) and egg phosphatidylglycerol (EPG) containing liposomes with or without cholesterol. The physical stability of the liposome dispersions was assessed by measuring the leak-in rate of a non-bilayer interacting hydrophilic marker molecule, calcein and changes in the particle size and its distribution in time. Additionally, permeability of either partially hydrolysed phospholipids or exogenous lyso-phosphatidylcholine(LPC) containing bilayers was calculated. The experiments were performed at 40 degrees C. Liposome dispersions were aged artificially by storing at 60 degrees C. The size of the liposomes and polydispersity index of the dispersions, in general, did not change significantly. The leak-in rate of calcein in externally added LPC containing liposomes was increased relative to the incorporated LPC concentration. The higher the LPC content of the bilayers, the higher the leak-in rate of calcein into liposomes. The leak-in rate of calcein, however, decreased first in partially hydrolysed phospholipids containing liposomes up to around 10% of hydrolysis and, afterwards, it started to increase. The leak-in rate was always lower in partially hydrolysed phospholipids containing liposomes than externally added LPC containing ones. Furthermore, the permeability of cholesterol containing bilayers was also always lower than the bilayers without cholesterol. In conclusion, addition of LPC into liposomal bilayers increases the permeability of bilayer. However, bilayers containing the hydrolysis products of phospholipids, both lyso-phospholipids and free fatty acids, did not show any enhanced permeability up to around 15% hydrolysis. Bilayer permeability is enhanced above 15% hydrolysis.     

New method for site-specific modification of liposomes with proteins using sortase A-mediated transpeptidation

A new method was developed for site-specific modifications of liposomes by proteins via sortase A (SrtA)-mediated transpeptidation reactions. In this regard, the enhanced green fluorescent protein (eGFP) was biologically engineered to carry at its polypeptide C-terminus the LPATG motif recognized by SrtA and used as the protein donor for linking to liposomes that were decorated with phospholipids carrying a diglycine motif as the other SrtA substrate and the eGFP acceptor. Under the influence of SrtA, eGFP was efficiently attached to liposomes, as proved by analyzing the enzymatic reaction products and the resultant fluorescent liposomes. It was observed that increasing the concentration and the distance of the diglycine motif on and from the liposome surface could significantly improve the efficiency of liposome modification by proteins. It is anticipated that this strategy can be widely useful for the modification of liposomes by other proteins.     

Interaction of liposomes with Zymomonas mobilis cells

Abstract Zymomonas mobilis (strain ZM4), an ethanoltolerant bacterium, interacts efficiently with liposomes consisting of Escherichia coli phospholipids. The parameters of the cell-liposome interaction have been determined. The process depends on temperature, presence of divalent cation, pH, liposome/cell ratio and is insensitive to pre-treatment of ZM4 cells with EDTA. Optimal transfer of exogenous phospholipids into cells was achieved at 37°C and pH 6.6, when the interaction medium contained 20 mM CaCl₂.     

Density-based separation of liposomes by glycerol gradient centrifugation

Sonicated liposomes of soybean phospholipids (asolectin) distribute nearly throughout a 19-22% (v/v) glycerol gradient when centrifuged to near equilibrium. Upon recentrifugation on an identical gradient, liposomes selected from several positions in such a gradient migrate as narrow bands to positions close to their original positions, indicating that the liposome distribution in the first gradient is the result of a density-based fractionation. Molecular sieve chromatography, turbidity, and trapped volume measurements indicate that the liposome densities are qualitatively related to their size, with the larger liposomes more dense than the smaller ones. Size estimates obtained by electron microscopy of negatively stained preparations indicate that the fractionation is effective for liposomes with diameters ranging from 200 to 600 A, with maximum efficiency in the range 200-300 A where the majority of the liposomes is found. Interestingly, high concentrations of liposomes improve the efficiency of the fractionation procedure. The size dependence of liposome density is shown not to be due to differential glycerol permeability or lipid composition, and is therefore most likely due to variations in the specific volumes of the individual phospholipid molecules owing to the curvature of the liposomes. Finally, freezing of the glycerol gradient fractions in liquid N2 and storage at -70 degrees C does not modify the size of the isolated liposomes. It is suggested that glycerol density gradient fractionation of liposomes could be a useful general method for obtaining liposomes of reasonably uniform size in large quantities and high concentrations.     

Characteristics of binding of zwitterionic liposomes to water-soluble proteins

The interactions of zwitterionic phospholipids phosphatidylcholine and phosphatidylethanolamine with protein proteinase inhibitors aprotinin and Bowman-Birk soybean proteinase inhibitor have been investigated. An increase in the hydrophobicity of the liposome surface was shown to be an important factor for the formation of proteoliposomes. According to ³¹P-NMR spectra, incorporation of the proteins into the liposomes does not influence the structural organization of the surface of the liposomes. Increasing the ionic strength does not inhibit the process of proteoliposome formation. Fluorescence assay of the complexes of anthracene-labeled phospholipids with the rhodamine B-labeled protein showed that after the encapsulation into the liposomes, the protein is located inside the particles and between the bilayers. Also, the effect of phospholipids with saturated fatty acid residues on the protein-lipid interaction was studied by differential scanning calorimetry. The results indicate that water-soluble proteins efficiently interact with zwitterionic phospholipids, and the encapsulation of the proteins into the liposomes is provided by electrostatic and hydrophobic forces (in the case of aprotinin) or predominantly by hydrophobic forces (Bowman-Birk soybean proteinase inhibitor).     

A method to determine the incorporation capacity of camptothecin in liposomes

The purpose of this study was to establish a new experimental approach to determine the maximum amount of campothecin (CPT) that can be incorporated in liposomes, and to use this method to compare the CPT-incorporation capacity of various liposome formulations. Small, CPT-saturated liposomes were prepared by dispersing freeze-dried blends of lipids and drug in phosphate buffer, and subsequent probe-sonication. Excess precipitated CPT could be separated from the liposomes by ultra-centrifugation. The small and homogeneous liposome size obtained gave a good and reproducible recovery of liposomes in the supernatant (>80%), whereas the acidic pH (pH 6.0) kept CPT in its hydrophobic lactone form, which is poorly soluble in the buffer. The maximum CPT-incorporation capacity of 12 different liposome formulations was investigated, using the described method, and was found to vary widely. With liposomes made of neutral and anionic phospholipids, the solubili ty of CPT in the buffer was improved by approximately a factor of 10 (from ∼2.7 to 15–50 μg/mL) as compared with buffer. With cationic liposomes containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a maximum CPT-solubilization of ∼100-fold, the buffer solubility was reached, probably owing to an electrostatic interaction between the cationic lipids and the carboxylate-CPT isomer. Increasing DOTAP fractions within egg-phosphatidylcholine (EPC)/DOTAP liposomes reached a CPT-incorporation plateau at ∼20 mol% DOTAP. The presented approach appears suitable to study the incorporation capacity of any drug component within small vesicles as long as the liposome incorporation is high relative to the intrisic water solubility of the drug.