HER2-Specific Affibody-Conjugated Thermosensitive Liposomes (Affisomes) for Improved Delivery of Anticancer Agents

Thermosensitive liposomes are attractive vehicles for the delivery and release of drugs to tumors. To improvethe targeting efficacy for breast cancer treatment, an 8.3-kDa HER2-specific Affibody molecule (Z_HER2:342-Cys) was conjugated to the surface of liposomes. The effects of this modification on physical characteristics and stability of the resulting nanoparticles denoted as “Affisomes” were investigated. Thermosensitive small unilamellar vesicle (SUV) liposomes of (80–100 nm) a diameter consisting of dipalmitoyl phosphatidylcholine (DPPC, Tm 41°C) as the matrix lipid and a maleimide-conjugated pegylated phospholipid (DSPE-MaL-PEG2000) were prepared by probe sonication. Fluorescent probes were incorporated into liposomes for biophysical and/or biochemical analysis and/or triggered-release assays. Affibody was conjugated to these liposomes via its C-terminal cysteine by incubation in the presence of a reducing agent (e.g., tributylphosphine) for 16–20 hours under an argon atmosphere. Lipid-conjugated affibody molecule was visible as an 11.3-kDa band on a 4–12% Bis/Tris gel under reducing conditions. Affibody conjugation yields were?～70% at a protein-lipid ratio of 20 μg/mg, with an average number of 200 affibody molecules per Affisome. Affibody conjugation to thermosensitive liposomes did not have any significant effect on the hydrodynamic size distribution of the liposomes. Thermosensitivity of Affisomes was determined by monitoring the release of entrapped calcein (a water-soluble fluorescent probe, λex/em 490/515 nm) as a function of temperature. Calcein was released from Affisomes (thermosensitive liposomes with affibody-Targeted SUV) as well as nontargeted SUV (thermosensitive liposomes without affibody) in a temperature-dependent manner, with optimal leakage (90–100%) at 41°C. In contrast, liposomes prepared from Egg phosphatidyl choline (Egg PC, Tm?～0°C) under similar conditions released only 5–10% calcein at 41°C. Affisomes, when stored at room temperature, retained?>?90% entrapped calcein up to 7 days. Moreover, incubation of liposomes in phosphate-buffered saline, supplemented with 10% heat-inactivated serum (fetal bovine serum) did not result in a destabilization of liposomes. Therefore, Affisomes present promising, novel drug-delivery candidates for breast cancer targeting.     

Gangliosides reduce leakage of aqueous-space markers from liposomes in the presence of human plasma

We have studied the role of glycolipids in reducing leakage of aqueous-space markers from liposomes, composed primarily of egg phosphatidylcholine, in the presence of human plasma. Liposomes were either small unilamellar (SUV) or large unilamellar (LUV). Leakage of liposome contents as affected by the incorporation into the liposomal bilayer of mono-, di-, or trisialogangliosides (GM, GD, GT) at different molar ratios in the presence or absence of cholesterol was examined. Leakage from liposomes decreased with increasing ganglioside sialic acid. Asialogangliosides had no effect on calcein leakage in the presence of plasma. The stabilizing effect of gangliosides and cholesterol was synergistic, and SUV containing 10 mol% GT and 33 mol% cholesterol had a half-life for leakage of calcein in plasma at 37 degrees C approaching 24 hours. LUV in the presence of plasma retained their contents longer than SUV, and gangliosides had an additional stabilizing effect. Phosphatidylserine and sulfatides were also capable of substituting for gangliosides in stabilizing liposomes to plasma-induced leakage. It appears that gangliosides stabilize liposomes in plasma at least in part through their ability to impart surface negative charge.     

Enzyme replacement via liposomes variations in lipid composition determine liposomal integrity in biological fluids

Liposomes survive exposure to biological fluids poorly, extruding trapped enzymes, drugs, or solutes upon interaction with serum or plasma constituents. We have quantified the disruptive effects of human serum on liposomes and have studied whether various modifications in their phospholipid composition might produce liposomes with an increased carrier potential for applications in vivo. Multilamellar liposomes (phosphatidylcholine 70:dicetyl phosphate 20: cholesterol 10) were prepared with ³H-labeled phosphatidylcholine as the lipid phase marker and [¹⁴C]inulin and horseradish peroxidase as aqueous phase markers. Gel exclusion chromatography showed that 32 ± 3% of [¹⁴C]inulin and 27 ± 7% of horseradish peroxidase were lost after 1 h incubation with 10% (v/v) human serum. Loss of aqueous solutes was reduced to 20 ± 5%/h and 17 ± 2%/h, respectively, after treatment with decomplemented serum (56°C, 30 min). Loss induced by serum was concentration and time dependent: to 57 ± 2% at 1 h and 67 ± 14% at 24 h, with 50% serum; plasma was slightly less perturbing whereas human serum albumin was not at all disruptive. By incorporating sphingomyelin (35 mol%) into multilamellar liposomes, the leakage of [¹⁴C]-inulin in the presence of 10% serum was reduced to 12 ± 4%/h; increasing the molar percentage of cholesterol to 35% also stabilized the lipid bilayers, reducing leakage to 20 ± 7%/h. Both small and large unilamellar vesicles could not be stabilized against serum-mediated leakage by the incorporation of sphingomyelin. The data suggest that cholesterol and sphingomyelin enhance liposomal integrity in the presence of serum or plasma and promise to yield enhanced survival of drug-laden lipid vesicles in biological fluids in vivo.     

HER2-specific affibody-conjugated thermosensitive liposomes (Affisomes) for improved delivery of anticancer agents

Thermosensitive liposomes are attractive vehicles for the delivery and release of drugs to tumors. To improvethe targeting efficacy for breast cancer treatment, an 8.3-kDa HER2-specific Affibody molecule (Z(HER2:342)-Cys) was conjugated to the surface of liposomes. The effects of this modification on physical characteristics and stability of the resulting nanoparticles denoted as "Affisomes" were investigated. Thermosensitive small unilamellar vesicle (SUV) liposomes of (80-100 nm) a diameter consisting of dipalmitoyl phosphatidylcholine (DPPC, Tm 41 degrees C) as the matrix lipid and a maleimide-conjugated pegylated phospholipid (DSPE-MaL-PEG2000) were prepared by probe sonication. Fluorescent probes were incorporated into liposomes for biophysical and/or biochemical analysis and/or triggered-release assays. Affibody was conjugated to these liposomes via its C-terminal cysteine by incubation in the presence of a reducing agent (e.g., tributylphosphine) for 16-20 hours under an argon atmosphere. Lipid-conjugated affibody molecule was visible as an 11.3-kDa band on a 4-12% Bis/Tris gel under reducing conditions. Affibody conjugation yields were approximately 70% at a protein-lipid ratio of 20 microg/mg, with an average number of 200 affibody molecules per Affisome. Affibody conjugation to thermosensitive liposomes did not have any significant effect on the hydrodynamic size distribution of the liposomes. Thermosensitivity of Affisomes was determined by monitoring the release of entrapped calcein (a water-soluble fluorescent probe, lambdaex/em 490/515 nm) as a function of temperature. Calcein was released from Affisomes (thermosensitive liposomes with affibody-Targeted SUV) as well as nontargeted SUV (thermosensitive liposomes without affibody) in a temperature-dependent manner, with optimal leakage (90-100%) at 41 degrees C. In contrast, liposomes prepared from Egg phosphatidyl choline (Egg PC, Tm approximately 0 degrees C) under similar conditions released only 5-10% calcein at 41 degrees C. Affisomes, when stored at room temperature, retained > 90% entrapped calcein up to 7 days. Moreover, incubation of liposomes in phosphate-buffered saline, supplemented with 10% heat-inactivated serum (fetal bovine serum) did not result in a destabilization of liposomes. Therefore, Affisomes present promising, novel drug-delivery candidates for breast cancer targeting.     

Phospholipase A(2)-catalyzed membrane leakage studied by immobilized liposome chromatography with online fluorescent detection

Unilamellar liposomes composed of phosphatidylcholine with an entrapped self-quenching fluorescent dye, calcein, were immobilized in chromatographic gel beads by avidin-biotin binding. Bee venom phospholipase A(2) (PLA(2)) was applied in a small amount onto the immobilized liposome column. The release of calcein from the immobilized liposomes resulting from the catalyzed hydrolysis of the phospholipids was detected online by immobilized liposome chromatography (ILC) using a flow fluorescent detector. The PLA(2)-catalyzed membrane leakage of the immobilized liposomes as studied with ILC was found to be affected by the gel pore size used for immobilization, by liposome size, and as expected by the concentration of calcium, but was unaffected by the flow rate of ILC. The largest PLA(2)-induced calcein release from the liposome column was detected on large unilamellar liposomes immobilized on TSK G6000PW or Sephacryl S-1000 gel in the presence of 1 mM Ca(2+) in the aqueous mobile phase. Comparison with the PLA(2)-catalyzed membrane leakage in free liposome suspensions, we conclude that the fluorescent leakage from liposomes hydrolyzed by PLA(2) can be rapidly and sensitively detected by ILC runs using large amount of immobilized liposomes with entrapped fluorescent dye.     

Temperature-dependent aggregation of pH-sensitive phosphatidyl ethanolamine-oleic acid-cholesterol liposomes as measured by fluorescent spectroscopy.

pH-sensitive liposomes made of phosphatidyl ethanolamine-oleic acid-cholesterol (4:2:4 molar ratio) at neutral pH values aggregate at approximately 40 degrees C. The aggregation is accompanied by liposome destabilization and by the release of intraliposomal fluorescent marker (calcein). Both aggregation and calcein leakage start at the temperature corresponding to the lipid phase transition into hexagonal phase. In the system studied the phase transition temperature interval is within 45 to 55 degrees C as estimated with the use of the fluorescent probe 1,6-diphenylhexatriene. The presence of cell cultivation medium RPMI 1640 decreases liposome aggregation temperature. The addition of 10% serum to the system decreases the temperature at which the aggregation proceeds still further. The conclusion that serum-free media should be used for cell experiments involving pH-sensitive liposomes is made.     

An increase in model lipid membrane fluidity as a result of local anesthetic action

The effect of local anesthetics on the permeability of phospholipid liposomes of different composition for calcein has been investigated. The local anesthetics tested included amides (lidocaine, prilocaine, mepivacaine, and bupivacaine) and esters (benzocaine, procaine, and tetracaine). The permeability of large monolamellar liposomes was assessed by monitoring the fluorescence of calcein leaking from the phospholipid vesicles. All tested amide anesthetics exerted negligible effects on the permeability of dioleylphosphocholine (DOPC) liposomes for the fluorescent marker. The most efficient in this group was did bupivacaine. Amides had a more pronounced effect on membranes in which 20 mol % of DOPC was replaced by tetraoleoylcardiolipin (TOCL). Benzocaine and procaine at concentration up to 100 mM did not affect the permeability of DOPC liposomes. Membrane permeability of DOPC liposomes was not affected by the addition of tetracaine to the final concentration of 2 mM, while the increase of anesthetic concentration up to 50 mM was accompanied by an increase in the intensity of fluorescence of calcein released from the vesicles, and addition of the anesthetic to the concentration of 100 mM caused by complete release of the marker incorporated by the liposomes. The threshold concentration of tetracaine initiating calcein leakage from vesicles that contained 20 mol % TOCL was 7 mM, and the concentration corresponding to 100% calcein leakage was 20 mM. Confocal fluorescence microscopy of giant monolamellar liposomes formed from an equimolar mixture of DOPC and tetramiristoylcardiolipin demonstrated the destruction of solid ordered domains at the presence of anesthetics, and its destructive capacity increasing in the following order: procaine ≈ mepivacaine < bupivacaine ? tetracaine. Variability of the depth of anesthetic incorporation into the membrane may account for the dissimilar effects of local anesthetics on liposomes.     

Spectroscopic and thermodynamic evidence for antimicrobial peptide membrane selectivity

In our laboratory we developed a series of antimicrobial peptides that exhibit selectivity and potency for prokaryotic over eukaryotic cells (Hicks et al., 2007). Circular dichroism (CD), isothermal calorimetry (ITC) and calcein leakage assays were conducted to determine the mechanism of lipid binding of a representative peptide 1 (Ac-GF-Tic-Oic-GK-Tic-Oic-GF-Tic-Oic-GK-Tic-KKKK-CONH₂) to model membranes. POPC liposomes were used as a simple model for eukaryotic membranes and 4:1 POPC:POPG liposomes were used as a simple model for prokaryotic membranes. CD, ITC and calcein leakage data clearly indicate that compound 1 interacts via very different mechanisms with the two different liposome membranes. Compound 1 exhibits weaker binding and induces less calcein leakage in POPC liposomes than POPC:POPG (4:1 mole ratio) liposomes. The predominant binding mechanism to POPC appears to be limited to surface interactions while the mechanism of binding to 4:1 POPC:POPG most likely involves some type of pore formation.     

Sendai virus induced leakage of liposomes containing gangliosides

Sendai virus induced liposome leakage has been studied by using liposomes containing a self-quenching fluorescent dye, calcein. The liposomes used in this study were prepared by a freeze and thaw method and were composed of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine (1:2.60:1.48 molar ratio) as well as various amounts of gangliosides and cholesterol. The leakage rate was calculated from the fluorescence increment as the entrapped calcein leaked out of the liposomal compartment and was diluted into the media. It was shown that the target liposome leakage was virus dose dependent. Trypsin-treated Sendai virus in which the F protein had been quantitatively removed did not induce liposome leakage, indicating that the leakage was a direct result of F-protein interaction with the target bilayer membrane. The activation energy of this process was approximately 12 kcal/mol below 17 degrees C and approximately 25 kcal/mol above 17 degrees C. Gangliosides GM1, GD1a, and GT1b could serve as viral receptor under appropriate conditions. Liposome leakage showed a bell-shaped curve dependence on the concentration of ganglioside in the liposomes. No leakage was observed if the ganglioside content was too low or too high. Inclusion of cholesterol in the liposome bilayer suppressed the leakage rate of liposomes containing GD1a. It is speculated that the liposome leakage is a consequence of fusion between Sendai virus and liposomes.     

Pore Formation by S. aureus α-toxin in Liposomes and Planar Lipid Bilayers: Effects of Nonelectrolytes

Nonelectrolytes such as polyethylene glycols (PEG) and dextrans (i) promote the association of S. aureus α-toxin with liposomes (shown by Coomassie staining) and (ii) enhance the rate and extent of calcein leakage from calcein-loaded liposomes; such leakage is inhibited by H⁺, Zn²⁺ and Ca²⁺ to the same extent as that of nonPEG-treated liposomes. Incubation of liposomes treated with α-toxin in the presence of PEG with the hydrophobic photo-affinity probe 3-(trifluoromethyl)-3-m-[¹²⁵I]iodophenyl)diazirine(¹²⁵I-TID) labels monomeric and—predominantly—hexameric forms of liposome-associated α-toxin; in the absence of PEG little labeling is apparent. At high concentrations of H⁺ and Zn²⁺ but not of Ca²⁺—all of which inhibit calcein leakage—the distribution of label between hexamer and monomer is perturbed in favor of the latter. In α-toxin-treated planar lipid bilayers from which excess toxin has been washed away, PEGs and dextrans strongly promote the appearance of ion-conducting pores. The properties of such pores are similar in most regards to pores induced in the absence of nonelectrolytes; they differ only in being more sensitive to ``closure' by voltage (as are pores induced in cells). In both systems, the stimulation by nonelectrolytes increases with concentration and with molecular mass up to a maximum around 2,000 Da. We conclude (i) that most of the α toxin that becomes associated with liposome or planar lipid bilayers does not form active pores and (ii) that the properties of α-toxin-induced pores in lipid bilayers can be modulated to resemble those in cells. Received: 2 October 1995/Revised: 3 November 1995     

Avidin–biotin-immobilized liposome column for chromatographic fluorescence on-line analysis of solute–membrane interactions

Unilamellar liposomes with entrapped fluorescent dye calcein were stably immobilized in gel beads by avidin–biotin-binding. The immobilized liposomes remained extremely stable upon storage and chromatographic runs. The immobilized calcein-entrapped liposomes were utilized for fluorescent analysis of solute–membrane interactions, which in some cases are too weak to be detected by chromatographic retardation. A liposome column was used as a sensitive probe to detect the interactions of membranes with pharmaceutical drugs, peptides and proteins. Retardation of the solutes was monitored using a UV detector. Perturbation of the membranes, reflected as leakage of the entrapped calcein by some of the solutes, can thus be detected on-line using a flow-fluorescent detector. For the amphiphilic drugs or synthetic peptides, perturbation of membranes became more pronounced when the retardation (hydrophobicity) of the molecules increased. On the other hand, in the case of positively-charged peptides, polylysine, or partially denatured bovine carbonic anhydrase, significant dye leakage from the liposomes was observed although the retardation was hardly to be measured. Weak protein–membrane interactions can thus be assumed from the large leakage of calcein from the liposomes. This provides additional useful information for solute–membrane interactions, as perturbation of the membranes was also indicated by avidin–biotin-immobilized liposome chromatography (ILC).     

Fusogenic activity of PEGylated pH-sensitive liposomes

The aim of this study was to investigate the fusogenic properties of poly(ethylene glycol) (PEG)ylated dioleoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) liposomes. These pH-sensitive liposomes were prepared by incorporating two different PEG lipids: distearoylphosphatidylethanolamine (DSPE)-PEG???? was mixed with the liposomal lipids using the conventional method, whereas sterol-PEG???? was inserted into the outer monolayer of preformed vesicles. Both types of PEGylated liposomes were characterized and compared for their entrapment efficiency, zeta potential and size, and were tested in vitro for pH sensitivity by means of proton-induced leakage and membrane fusion activity. To mimic the routes of intracellular delivery, fusion between pH-sensitive liposomes and liposomes designed to simulate the endosomal membrane was studied. Our investigations confirmed that DOPE/CHEMS liposomes were capable of rapidly releasing calcein and of fusing upon acidification. However, after incorporation of DSPE-PEG???? or sterol-PEG???? into the membrane, pH sensitivity was significantly reduced; as the mol ratio of PEG-lipid was increased, the ability to fuse was decreased. Comparison between two different PEGylated pH-sensitive liposomes showed that only vesicles containing 0.6 mol% sterol-PEG???? in the outer monolayer were still capable of fusing with the endosome-like liposomes and showing leakage of calcein at pH 5.5.     

Temperature-controlled interaction of thermosensitive polymer-modified cationic liposomes with negatively charged phospholipid membranes

To obtain cationic liposomes of which affinity to negatively charged membranes can be controlled by temperature, cationic liposomes consisting of 3beta-[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol and dioleoylphosphatidylethanolamine were modified with poly(N-acryloylpyrrolidine), which is a thermosensitive polymer exhibiting a lower critical solution temperature (LCST) at ca. 52 degrees C. The unmodified cationic liposomes did not change its zeta potential between 20-60 degrees C. The polymer-modified cationic liposomes revealed much lower zeta potential values below the LCST of the polymer than the unmodified cationic liposomes. However, their zeta potential increased significantly above this temperature. The unmodified cationic liposomes formed aggregates and fused intensively with anionic liposomes consisting of egg yolk phosphatidylcholine and phosphatidic acid in the region of 20-60 degrees C, due to the electrostatic interaction. In contrast, aggregation and fusion of the polymer-modified cationic liposomes with the anionic liposomes were strongly suppressed below the LCST. However, these interactions were enhanced remarkably above the LCST. In addition, the polymer-modified cationic liposomes did not cause leakage of calcein from the anionic liposomes below the LCST, but promoted the leakage above this temperature as the unmodified cationic liposomes did. Temperature-induced conformational change of the polymer chains from a hydrated coil to a dehydrated globule might affect the affinity of the polymer-modified cationic liposomes to the anionic liposomes.     

Bilayer membrane destabilization induced by dolichylphosphate

Small vesicles containing the fluorescent probe calcein were used to investigate the effect of dolichyl phosphate (Dol-P) on phospholipid bilayer stability. In the absence of Dol-P, phospholipid vesicles retained the fluorescent probe upon the addition of divalent cations. Small vesicles containing Dol-P, however, exhibited calcein leakage when incubated in the presence of divalent cations. This effect was observed in liposomes composed of a mixture of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and Dol-P, but not in PC/Dol-P liposomes. The rate of calcein leakage was proportional to divalent cation concentration and to temperature, but was independent of vesicle concentration. These results demonstrate that Dol-P has significant effects on the stability of PE containing phospholipid bilayers. Vesicle leakage was also promoted by the addition of rat liver Dol-P-mannose synthase (EC 2.4.1.83) to intact PE/PC/Dol-P vesicles. Enzyme induced leakage from phospholipid vesicles required the presence of both unsaturated PE and Dol-P. The phospholipid composition of leaky vesicles could be correlated with the lipid matrix required for maximal transferase activity of the rat liver synthase. The destabilizing effects of Dol-P on phospholipid bilayers may therefore be involved in the translocation of activated sugars across biological membranes.     

Temperature-dependent permeability of large unilamellar liposomes

The temperature-dependent drug leakage from liposomes composed of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol (4:1, by weight) was studied. Experiments were performed in Hepes buffer and 50% fetal calf serum. Large unilamellar liposomes were formed by the reverse phase evaporation process and extruded through a series of polycarbonate membranes with pore sizes of 0.4, 0.2, 0.1 and 0.08 μm. The release of the water soluble radioisotopes cytosine 1-β-D-[³H]arabinofuranoside and [³H]inulin from the aqueous compartment of these liposomes was measured as a function of time and temperature. Both radioisotopes were released at temperatures near 42°C, the solid-to-liquid-crystalline phase transition temperature of these lipids. The percent drug release decreased as the size of the liposomes was reduced. This effect was more pronounced in Hepes buffer than serum. The release of both radioisotopes was greatest at 40°C in Hepes buffer and at 43°C in 50% fetal calf serum. In addition, the rate of drug release was much faster in serum than in buffer. These results suggest that different drug release processes are occurring in buffer and in serum.     

The influence of the internal content of negatively charged liposomes on their interaction with high-density lipoprotein

The release of the internal content of negatively charged phosphatidylcholine/phosphatidylserine vesicles under the influence of high density lipoprotein was studied. Under standard conditions (the same composition outside and inside the compartment) the leakage of negative liposomes increased significantly. However, a high internal concentration of calcein provoked a sealing effect, exhibited both in sucrose and in calcein release. This sealing effect is not related to the size of vesicles, the fluidity of the membrane, the distribution of phosphatidylserine molecules, or the membrane potential. Our data indicate that surface potential influences this effect, probably in addition to a lateral pressure effect such as with cholesterol. The surface potential, as measured by the water-lipid partition coefficient of fatty acids, is strongly affected by internal ionic strength when liposomes contain calcein as well as other polyanions (6-carboxyfluorescein, sodium citrate).     

Improved Cytoplasmic Delivery to Plant Protoplasts via pH-Sensitive Liposomes

We demonstrated that the liposomes composed of dioleolylphosphatidylethanolamine/cholesterol/oleic acid (4:4:2) dramatically release their contents at a pH of less than or equal to 6.0 and are capable of delivering their contents into the cytoplasm of higher plant protoplasts. This is shown by using a soluble fluorescent dye, calcein, as a liposome-entrapped marker. We found that calcein fluorescence was evenly distributed in the cytoplasm of wild carrot protoplasts after the incubation of protoplasts with liposomes in the presence of polyethylene glycol 6000. At 0.45 micro mole phospholipid per 6 × 10⁵ protoplast, for example, the percentage of protoplasts which took up liposomes was 89% which was much higher than that achieved by conventional pH-insensitive liposomes. In this study, liposomes were prepared by a detergent dialysis method which avoided sonication and organic solvents. Thus macromolecules such as proteins and nucleic acids could be entrapped in the liposomes and delivered to the cytoplasm of the protoplasts.     

Enzyme replacement via liposomes. Variations in lipid compositions determine liposomal integrity in biological fluids.

Liposomes survive exposure to biological fluids poorly, extruding trapped enzymes, drugs, or solutes upon interaction with serum or plasma constituents. We have quantified the disruptive effects of human serum on liposomes and have studied whether various modifications in their phospholipid composition might produce liposomes with an increased carrier potential for application in vivo. Multilamellar liposomes (phosphatidycholine 70:dicetyl phosphate 20:cholesterol 10) were prepared with 3H-labeled phosphatidylcholine as the lipid phase marker and [14C]inulin and horseradish peroxidase as aqueous phase markers. Gel exclusion chromatography showed that 32 +/- 3% of [14C]inulin and 27 +/- 7% of horseradish peroxidase were lost after 1 h incubation with 10% (v/v) human serum. Loss of aqueous solutes was reduced to 20 +/- 5%/h and 17 +/- 2%/h, respectively, after treatment with decomplemented serum (56 degrees C, 30 min). Loss induced by serum was concentration and time dependent: to 57 +/- 2% at 1 h and 67 +/- 14% at 24 h, with 50% serum; plasma was slightly less perturbing whereas human serum albumin was not at all disruptive. By incorporating sphingomyelin (35 mol%) into multilamellar liposomes, the leakage of [14c]-inulin in the presence of 10% serum was reduced to 12 +/- 4%/h; increasing the molar percentage of cholesterol to 35% also stabilized the lipid bilayers, reducing leakage to 20 +/- 7%/h. Both small and large unilamellar vesicles could not be stablilized against serum-mediated leakage by the incorporation of sphingomyelin. The data suggest that cholesterol and sphingomyelin enhance liposomal integrity in the presence of serum or plasma and promise to yield enhanced survival of drug-laden lipid vesicles in biological fluids in vivo.     

Small, but not large, unilamellar liposomes composed of dioleoylphosphatidylethanolamine and oleic acid can be stabilized by human plasma

Small unilamellar liposomes, composed of dioleoylphosphatidylethanolamine (DOPE) and oleic acid (OA), prepared by sonication, were incubated in the presence of human plasma at 37 degrees C. The release of entrapped calcein after 8-h incubation was about 15% in plasma, compared with about 70% in phosphate-buffered saline under the same conditions. In contrast, dioleoylphosphatidylcholine (DOPC)/OA liposomes under the same conditions release about 70% in plasma and only 10% in PBS. Total release of calcein from the DOPE/OA liposomes was observed in a PBS solution containing bovine serum albumin, and the release was completely blocked by preincubation of the liposomes with plasma. These results indicate that the unstable DOPE/OA liposomes are stabilized by incubation with plasma. The stabilization process was very fast, being completed within 1 min. Only relatively small liposomes (d less than or equal to 200 nm) were completely stabilized by plasma; larger liposomes were progressively less stabilizable. SDS-polyacrylamide gel electrophoresis of liposomes which had been incubated with plasma and then washed indicated that several proteins were tightly associated with liposomes. Using liposomes containing [14C]OA, it was found that about 70% of the original OA was extracted after 1-h incubation with human plasma at 37 degrees C. Thin-layer chromatographic analysis of the plasma-treated liposomes showed the presence of the plasma lipids in the liposomes. These results suggest that liposomes composed of DOPE/OA are stabilized by protein and/or lipid components from human plasma and that the composition of the liposomes is altered. The mechanism of stabilization is discussed in terms of the surface pressure of small vesicles with a high degree of curvature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trypsin induced destabilization of liposomes composed of dioleoylphosphatidylethanolamine and glycophorin

Destabilization of liposomes composed of phosphatidylethanolamine (PE) and purified glycophorin of human erythrocytes was studied with the release of an entrapped fluorescent dye, calcein. Proteolytic cleavage of liposomes by trypsin induced a rapid increase of turbidity and the leakage of calcein from the liposomes. Kinetic experiments indicated that the destabilization was a second order reaction, i.e. it required liposome collision. Using N-(7-nitro-2,1,3-benzoxadiazol-4-yl) PE as a fluorescent probe for the formation of hexagonal phase of PE, tryptic digestion of the liposomes resulted in a higher tendency of the PE bilayer to transform into the hexagonal phase. We propose that hexagonal (or inverted micellar) structures are involved in the trypsin induced liposome destabilization.