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.     

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.     

Interaction of liposomes with Kupffer cells in vitro

We investigated the interaction of liposomes with rat Kupffer cells in monolayer maintenance culture. The liposomes (large unilamellar vesicles, LUV) were composed of 14C-labelled phosphatidylcholine, cholesterol and phosphatidylserine (molar ratio 4:5:1) and contained either 3H-labelled inulin or 125I-labelled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. After 2-3 days in culture the cells exhibited optimal uptake capacity. The uptake process showed saturation kinetics, maximal uptake values amounting to 2 nmol of total liposomal lipid/h/10(6) cells. This is equivalent to 1500 vesicles per cell. The presence of fetal calf serum (FCS) during incubation increased uptake nearly two-fold, whereas freshly isolated rat serum had no effect. The binding of the liposomes to the cells caused partial release of liposomal contents (about 15-20%) both at 4 degrees C and at 37 degrees C. In the presence of metabolic inhibitors the uptake at 37 degrees C was reduced to about 20% of the control values. Inulin and lipid label became cell-associated at similar rates and extents, whereas the association of albumin label gradually decreased after attaining a maximum at relatively low values. When, after 1 h incubation, the liposomes were removed continued incubation for another 2 h in absence of liposomes led to an approx. 30% release of cell-associated lipid label into the medium in water-soluble form. Under identical conditions as much as 90% of the cell-associated albumin label was released in acid-soluble form. Contrarily, the inulin label remained firmly cell-associated under these conditions. From these results we conclude that Kupffer cells in monolayer culture take up liposomes primarily by way of an adsorptive endocytic mechanism. This conclusion was confirmed by morphological observations on cells incubated with liposomes containing fluorescein isothiocyanate (FITC) dextran or horseradish peroxidase as markers for fluorescence microscopy and electron microscopy, respectively.     

Isolation and low-temperature preservation of liposomes containing rifampicin

The optimal conditions for preparations of rifampicin-containing liposomes were determined with the methods of mechanical shaking, gas dispersion and and reversible phases. It was found that the percentage of rifampicin incorporation into liposomes depended on the molar ratio of the antibiotic to the lipid (the optimal ratio was 1 : 10), the size and structure of liposomes, the amount of cholesterol added and the lipid membrane charge. Incorporation of rifampicin amounted to 16.1 +/- 2.4, 39.2 +/- 3.2 and 60.5 +/- 2.9 per cent with respect to neutral lecithin multilamellar liposes, liposomes prepared with the gas dispersion method and liposomes prepared with the method of reversible phases, respectively. Cholesterol in a molar ratio to lecithin equal to 2 : 5 or higher and dicetyl phosphate imparting the negative charge to the membrane had an inhibitory effect on the drug uptake by liposomes, while stearyl amine having the positive charge had a stimulating effect. The effect of the cryoprotectors glucose, polyvinylpyrrolidone, poly-ethylene glycole-400 and glycerol on low-temperature preservation and storage of rifampicin-containing liposomes was studied. It was shown that 10--15 per cent solutions of sucrose and glucose had the highest cryoprotective effect, when the two-stage method of freezing was used. It provided almost 84 per cent preservation of liposomal rifampicin. Electron microscopy showed that after defrosting liposomes no significant changes in the size and structure of lipid membranes were observed.     

Intracellular digestion of saturated and unsaturated phospholipid liposomes by mucosal cells. Possible mechanism of transport of liposomally entrapped macromolecules across the isolated vascularly perfused rabbit ileum

The mechanism of intestinal absorption of liposomally entrapped [14C]inulin and 125I-labelled poly(vinylpyrrolidone) was studied using the isolated rabbit intestinal loop with intact perfused vasculature, a system more closely resembling an in vivo system than the everted sac technique. [14C]Inulin or 125I-poly(vinylpyrrolidone) was entrapped in liposomes prepared from unsaturated egg phosphatidylcholine and soya phosphatidylcholine, and saturated distearoylphosphatidylcholine (18:0), dipalmitoylphosphatidylcholine (16:0) and dimyrostoylphosphatidylcholine (14:0). Free and liposomally entrapped macromolecules were introduced in the ileum and the transport of liposomes and entrapped macromolecules into the venous effluent was monitored by measuring the presence of the aqueous marker 125I-poly(vinylpyrrolidone) or [14C]inulin, and lipid marker [3H]cholesterol. The results show that intact liposomes are not transported across intestine into the venous effluent, but they are taken up by mucosal cells and digested intracellularly, releasing the entrapped markers 125I-poly(vinylpyrrolidone) and [14C]inulin. These markers are then transported into the venous effluent as free molecules. The absorption of liposomally entrapped [14C]inulin into the venous effluent is biphasic, first slow for 30 min (i.e., a lag period of 30 min), followed by a rapid linear increase. The duration of the lag period and the rate of absorption of the entrapped [14C]inulin are dependent on the degree of saturation and the transition temperature of the phospholipids used to prepare liposomes. The possible explanation of the lag period based on the evidence presented here is that it is the time required for the liposomes to be taken up by mucosal cells and digested intracellularly. Intracellular digestion of liposomes prepared from saturated phospholipids is more rapid than from those prepared from unsaturated phospholipids, and the greater the fatty acid chain length of the saturated phospholipids the more rapid the intracellular degradation of liposomes.     

Liposome-mediated transfer of macromolecules into flagellated cell envelopes from bacteria

We have studied the interaction between flagellated cell envelopes from Escherichia coli and liposomes. Oligolamellar liposomes of ca. 0.45-micron diameter, composed of azolectin, phosphatidylserine, and cholesterol at a molar ratio of 7:1:2, were prepared by freezing and thawing and subsequent extrusion through polycarbonate filters. These liposomes exhibited high entrapment capacity and low leakiness. Liposome-cell envelope interaction was monitored flow cytometrically in a fluorescence-activated cell sorter with a fluorescent aqueous space marker and by a filtration assay with radiolabels for the lipid phase and the liposomal aqueous space. Maximal association of liposomes with the envelopes was observed in both assays after ca. 25 min at 30 degrees C. After such period of time, it seems that up to 200 liposomes (depending on the liposome to envelope ratio) were associated with a single cell envelope, as calculated from the radiotracer studies. Fluorometric measurements of the transfer of liposomal contents and the intermixing of membrane lipids indicated that at least 20% of the envelope-associated liposomes had delivered their content into the envelopes, possibly by fusion. Electron microscopic observations confirmed the transfer of liposome-encapsulated ferritin molecules into the cell envelopes. Our data suggest that liposomal carriers might be employed to deliver cytoplasmic, chemotaxis-related macromolecules into bacterial cell envelopes.     

Effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of liposomes by Kupffer cells in culture

We investigated the effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of large unilamellar liposomes by rat Kupffer cells in maintenance culture. The phospholipid vesicles were labeled in the lipid moiety with phosphatidyl[14C]choline and contained [3H]inulin or [125I]iodoalbumin as nondegradable and degradable markers of the aqueous vesicle content, respectively. Cytochalasin B and dihydrocytochalasin B, inhibitors of microfilament function, reduced inert inulin label uptake by 75% maximally, but residual uptake was not followed by release of lipid degradation products from the cells. By contrast, colchicine, an inhibitor of microtubule assembly, reduced uptake of liposomal inulin by maximally 55% but could not inhibit release of lipid degradation products from the cells. It is concluded that the cytochalasins partly inhibit uptake but fully prevent the arrival of internalized liposomes in the lysosomal compartment, while the action of colchicine is to slow down the overall process of uptake and subsequent transportation to the lysosomes. Monensin reduced inulin uptake to an extent similar to that found with colchicine, but reversibly blocked degradation of liposomal lipid and encapsulated protein. The kinetics of degradation of liposomal constituents suggests that residual uptake in the presence of monensin represents accumulation in an intracellular compartment. Trifluoperazine did not affect binding, internalization or degradation of encapsulated protein at low concentration (6 microM), but completely inhibited release of liposomal lipid degradation products under these conditions. At intermediate concentration (14 microM), the drug also reduced the internalization, while a high concentration (22 microM) was required to inhibit protein degradation as well. We conclude that trifluoperazine has multiple sites of action in the uptake and processing of liposomal constituents by Kupffer cells.     

Properties of stearylamine liposomes containing tyrosine phenol-lyase

The activity of tyrosine phenol-layse a chemotherapeutic enzyme with a dissociable pyridoxal phosphate cofactor, was studied after incorporation into multilamellar positively charged liposomes. Tyrosine phenol-lyase activity was assessed in the presence and absence of exogenous pyridoxal phosphate. A maximum of 75% total enzyme activity was associated with liposomes when prepared from a molar lipid ratio of egg lecithin, cholesterol, stearylamine (7 : 2 : 1, w/w). The total tyrosine phenol-lyase activity was comprised of 25% membrane-associated enzyme and 50% encapsulated enzyme. Encapsulation increased the stability of the enzyme under the in vitro conditions of cold storage at 4°C for 3 weeks and under elevated temperatures up to 61°C. Liposomal encapsulation afforded little protection against trypsin and no protection against whole mouse plasma in vitro. Heat-treated plasma (100°C for 1 h) had little effect on the activity of free and encapsulated tyrosine phenol-lyase. These results indicated that whole plasma contained a heat-labile factor(s) which destroyed both the liposomal and free tyrosine phenol-lyase activity. Plasma clearance after intraperitoneal injection of tyrosine phenol-lyase in B6D2F₁ female mice was reduced by liposomal encapsulation, particularly when the animals were pre-treated with empty liposomes; however, only a small proportion of free and liposomal tyrosine phenol-lyase was absorbed. The free enzyme rapidly lost holoenzyme activity after absorption but the liposomes maintained holoenzyme activity. Even though liposomes preserved holo-tyrosine phenol-lyase activity, the holoenzyme was not present in sufficient concentration to sustain a reduced plasma tyrosine level.     

Interaction of digitonin and its analogs with membrane cholesterol

The interaction of digitonin with membrane cholesterol was studied by using various digitonin analogs, and radioactive desglucodigitonin. The following results were obtained concerning the effect of digitonin on erythrocytes, granulocytes and liposomes. Digitonin and its analogs showed activity to induce hemolysis, granulocyte activation and liposomal membrane damage. The activity was affected by change of the carbohydrate residue of the molecule; the order of hemolytic activity was digitonin greater than or equal to desglucodigitonin much greater than glucosyl-galactosyl-digitogenin greater than galactosyl-digitogenin, digitogenin. The relative activities of these compounds to induce granulocyte activation and liposomal membrane damage were similar to those observed in the hemolysis. [3H]Desglucodigitonin could bind to cholesterol in liposomes. The binding was stoichiometric and the ratio of desglucodigitonin bound to liposomes/cholesterol in liposomes was close to 1, irrespective of the cholesterol content in liposome. Damage to liposomes was, however, induced by desglucodigitonin only when they contained more than 0.2 molar ratio of cholesterol to phospholipid. Addition of digitonin as well as desglucodigitonin to preformed liposomes deprived of cholesterol affected the anisotropic molecular motion of spin-labeled phosphatidylcholine incorporated into the liposomes, suggesting that the molecules could be inserted into the lipid bilayer free of cholesterol. Molecules of desglucodigitonin in the lipid phase may, however, be equilibrated with those in the aqueous phase, unless they form a complex with cholesterol, since no appreciable amount of [3H]desglucodigitonin could be detected in the liposome fraction after separation by column chromatography. Digitonin decreased the order parameter of spin-labeled phosphatidylcholine when liposomes contained equimolar cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Encapsulation of Chicken Egg Yolk Immunoglobulin G (IgY) by Liposomes

Encapsulation of antibodies isolated from chicken egg yolk (IgY) in egg lecithin/cholesterol liposomes was attempted. IgY was successfully encapsulated into the liposomes by using the dehydration-rehydration method. Electron microscopic observation demonstrated that the liposomes prepared by this method were large multilamellar vesicles with a diameter of several μm. The encapsulation efficiency was improved by increasing the rehydration temperature to 60°C. The cholesterol/lecithin ratio also affected the efficiency, giving the highest value at a ratio of 1/4 (mol/mol). Some efflux of glucose through the liposomal membranes was observed, particularly for the liposome with a low cholesterol content, but that of IgY was not detected, irrespective of the cholesterol content. Encapsulation reduced the activity loss of the IgY antibodies under acidic conditions. IgY encapsulated in the liposomes was also markedly resistant to pepsin hydrolysis, which usually results in complete loss of activity with unencapsulated IgY, suggesting that liposomal encapsulation is an effective means for protecting IgY under gastric conditions.     

Relation between the prolongation of the anti-inflammatory activity of liposome-encapsulated hydrocortisone and liposome composition in experimental arthritis

Experiments on rabbits with arthritis have demonstrated the possibility of a 10-fold decrease in the dose of hydrocortisone acetate incorporated into liposomes, administered intraarticularly as compared with a commercial drug in the form of suspension. The antiinflammatory effect was found to be appreciably prolonged (up to 5-6 days) upon the use of dipalmitoylphosphatidylcholine liposomes with 20 mol% cholesterol. Hydrocortisone had a prolongation effect (about 1-2 days) in the lipid phase of multilamellar liposomes from egg lecithin, dimyristoylphosphatidylcholine and distearoylphosphatidylcholine.     

Effects of Triton X-100 concentration and incubation temperature on carboxyfluorescein release from multilamellar liposomes

Carboxyfluorescein is the most commonly used probe to measure the rate of release of vesicle contents. The validity of the data obtained by this method depends on obtaining an end point based on the complete release of the dye on treatment of the liposomes with a detergent, usually Triton X-100. However, Triton does not completely release entrapped carboxyfluorescein from multilamellar liposomes and the amount and rate of release of marker upon detergent treatment is a function of lipid composition of the liposome, Triton concentration and temperature and duration of detergent incubation. The fluorescence ‘end point’ for distearoyl-l-α-phosphatidylcholine/cholesterol (2:1, mol%) multilamellar liposomes treated with 0.5% Triton at 22°C (a condition often used) is only about one-fifth the value for liposomes treated with 5% Triton at 72°C. The conditions of treatment appear to affect the release of carboxyfluorescein from the lipid of the partially or completely disrupted liposome and the subsequent partitioning of the free dye into the aqueous phase. This effect can lead to serious errors in the interpretation of multilamellar liposome stability data. However, Triton allows complete release of entrapped dye from small unilamellar vesicles under all conditions tested.     

Interaction of steroids with adrenal cytochrome P-450 (P-450(17)alpha,lyase) in liposome membranes

Purified cytochrome P-450(17)alpha,lyase from guinea-pig adrenal microsomes, which catalyzes progesterone 17 alpha-hydroxylation and sequentially C17-C20 bond cleavage of the 17 alpha-hydroxyprogesterone, was successfully incorporated into liposomal membranes composed of only phosphatidylcholine or of a phospholipid mixture of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine at a molar ratio of 5:3:1. Although the purified P-450(17)alpha,lyase was readily converted into P-420 in the detergent-solubilized system without substrates, the P-450 embedded in the liposomal membranes was found to be quite stable without the substrates. Using the P-450(17)alpha,lyase-proteoliposomes, the interaction of steroids with P-450(17)alpha,lyase was studied for progesterone, 17 alpha-hydroxyprogesterone and androstenedione in the liposomal system by optical difference spectroscopy and by equilibrium dialysis. The partition coefficients of steroids between the aqueous phase and the liposomal membranes were determined by the equilibrium dialysis. They were about 1.4-1.6-times higher in phosphatidylcholine liposomes than in the liposomes of the lipid mixture. The dissociation constants of the P-450-steroid complexes were calculated from the apparent dissociation constants using the partition coefficients for the situation where the substrate-binding site faces the lipid phase of the membranes or where it faces the aqueous phase. The dissociation constant in the former case was not affected by the lipid composition. These results suggest that P-450(17)alpha,lyase might interact only with the substrates in the lipid phase of the liposomal membranes.     

Permeability changes induced by 130 GHz pulsed radiation on cationic liposomes loaded with carbonic anhydrase

The effects of pulsed 130 GHz radiations on lipid membrane permeability were investigated by using cationic liposomes containing dipalmitoyl phosphatidylcholine (DPPC), cholesterol, and stearylamine. Carbonic anhydrase (CA) was loaded inside the liposomes and the substrate p-nitrophenyl acetate (p-NPA) added in the bulk aqueous phase. Upon permeation across the lipid bilayer, the trapped CA catalyzes the conversion of the p-NPA molecules into products. Because the self-diffusion rate of p-NPA across intact liposomes is very low the CA reaction rate, expressed as Delta A/min, is used to track membrane permeability changes. The effect of 130 GHz radiation pulse-modulated at low frequencies of 5, 7, or 10 Hz, and at time-averaged incident intensity (I(AV)) up to 17 mW/cm(2) was studied at room temperature (22 degrees C), below the phase transition temperature of DPPC liposomes. At all the tested values of I(AV) a significant enhancement of the enzyme reaction rate in CA-loaded liposomes occurred when the pulse repetition rate was 7 Hz. Typically, an increase from Delta A/min = 0.0026 +/- 0.0010 (n = 11) to Delta A/min = 0.0045 +/- 0.0013 (n = 12) (P < 0.0005) resulted at I(AV) = 7.7 mW/cm(2). The effect of 130 GHz pulse-modulated at 7 Hz was also observed on cationic liposomes formed with palmitoyloleoyl phosphatidylcholine (POPC), at room temperature (22 degrees C), above the phase transition temperature of POPC liposomes.     

Effect of the cholesterol content of small unilamellar liposomes on their stability in vivo and in vitro

Small unilamellar neutral, negatively and positively charged liposomes composed of egg phosphatidylcholine, various amounts of cholesterol and, when appropriate, phosphatidic acid or stearylamine and containing 6-carboxyfluorescein were injected into mice, incubated with mouse whole blood, plasma or serum or stored at 4°C. Liposomal stability, i.e. the extent to which 6-carboxyfluorescein is retained by liposomes, was dependent on their cholesterol content. (1) Cholesterol-rich (egg phosphatidylcholine/cholesterol, 7:7 molar ratio) liposomes, regardless of surface charge, remained stable in the blood of intravenously injected animals for up to at least 400min. In addition, stability of cholesterol-rich liposomes was largely maintained in vitro in the presence of whole blood, plasma or serum for at least 90min. (2) Cholesterol-poor (egg phosphatidylcholine/cholesterol, 7:2 molar ratio) or cholesterol-free (egg phosphatidylcholine) liposomes lost very rapidly (at most within 2min) much of their stability after intravenous injection or upon contact with whole blood, plasma or serum. Whole blood and to some extent plasma were less detrimental to stability than was serum. (3) After intraperitoneal injection, neutral cholesterol-rich liposomes survived in the peritoneal cavity to enter the blood circulation in their intact form. Liposomes injected intramuscularly also entered the circulation, although with somewhat diminished stability. (4) Stability of neutral and negatively charged cholesterol-rich liposomes stored at 4°C was maintained for several days, and by 53 days it had declined only moderately. Stored liposomes retained their unilamellar structure and their ability to remain stable in the blood after intravenous injection. (5) Control of liposomal stability by adjusting their cholesterol content may help in the design of liposomes for effective use in biological systems in vivo and in vitro.     

Spectrophotometric measurement of intraliposomal trehalose

Trehalose, a non-reducing glucose disaccharide found at high concentrations in many species of anhydrobiotic organisms, shows significant promise in protecting cellular viability and structural integrity during freezing and desiccation. As mammalian cell membranes are impermeable to trehalose, extensive efforts have been taken to introduce trehalose into mammalian cells. In this study, we report on the characterization of trehalose-containing liposomes, with focus on the entrapment of trehalose inside liposomes, as the first step in establishing liposomes as a delivery system in the biopreservation field. Liposomes were synthesized by hydrating a phospholipid/cholesterol lipid bilayer with 200-400 mM trehalose buffer and repeatedly extruding the lipid suspension to form unilamellar vesicles. The trehalose content of the liposomal lysate was determined spectrophotometrically using a commercial kit Megazyme and confirmed with HPLC measurements. The number of liposomes was calculated from the phosphate content of the liposomal preparation and an estimated number of lipid molecules in a 401+/-8 nm liposome. Based on an intraliposomal trehalose content, the calculated liposomal encapsulation efficiency of 200 mM trehalose liposomes was of 92+/-0.7%. This value was in agreement with the 300 and 400 mM trehalose liposomes (91.1+/-8.2% and 102.1+/-9.4%, respectively). The Megazyme method for trehalose measurement is an inexpensive and sensitive technique that does not require specialized instrumentation or extensive technical expertise. Therefore, it can be used to enhance current efforts in the development of alternative strategies for the cryo- and lyoprotection of mammalian cells.     

A novel method to label preformed liposomes with 64Cu for positron emission tomography (PET) imaging

Radiolabeling of liposomes with 64Cu (t(1/2)=12.7 h) is attractive for molecular imaging and monitoring drug delivery. A simple chelation procedure, performed at a low temperature and under mild conditions, is required to radiolabel preloaded liposomes without lipid hydrolysis or the release of the encapsulated contents. Here, we report a 64Cu postlabeling method for liposomes. A 64Cu-specific chelator, 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N',N',N'-tetraacetic acid (BAT), was conjugated with an artificial lipid to form a BAT-PEG-lipid. After incorporation of 0.5% (mol/mol) BAT-PEG-lipid during liposome formulation, liposomes were successfully labeled with 64Cu in 0.1 M NH4OAc pH 5 buffer at 35 degrees C for 30-40 min with an incorporation yield as high as 95%. After 48 h of incubation of 64Cu-liposomes in 50/50 serum/PBS solution, more than 88% of the 64Cu label was still associated with liposomes. After injection of liposomal 64Cu in a mouse model, 44+/-6.9, 21+/-2.7, 15+/-2.5, and 7.4+/-1.1 (n=4) % of the injected dose per cubic centimeter remained within the blood pool at 30 min, 18, 28, and 48 h, respectively. The biodistribution at 48 h after injection verified that 7.0+/-0.47 (n=4) and 1.4+/-0.58 (n=3) % of the injected dose per gram of liposomal 64Cu and free 64Cu remained in the blood pool, respectively. Our results suggest that this fast and easy 64Cu labeling of liposomes could be exploited in tracking liposomes in vivo for medical imaging and targeted delivery.     

Liposomes with entrapped doxorubicin exhibit extended blood residence times

The blood residence time of liposomes with entrapped doxorubicin is shown to be significantly longer than for identically prepared empty liposomes. Liposomal doxorubicin systems with a drug-to-lipid ratio of 0.2 (w/w) were administered at a dose of 100 mg lipid/kg. Both doxorubicin and liposomal lipid were quantified in order to assess in vivo stability and blood residence times. For empty vesicles composed of phosphatidylcholine (PC)/cholesterol (55:45, mole ratio) and sized through filters of 100 nm pore size, 15-25% of the administered lipid dose was recovered in the blood 24 h after i.v. injection. The percentage of the dose retained in the circulation at 24 h increased 2-3-fold when the liposomes contain entrapped doxorubicin. For 100 nm distearoyl PC/chol liposomal doxorubicin systems, as much as 80% of the injected dose of lipid and drug remain within the blood compartment 24 h after i.v. administration.     

Preparation of liposomes containing lysosomal enzymes for therapeutic use

The preparation of fused materials using liposomes has been examined for several decades as a tool for the stabilization of heterogeneous enzymes. We investigated the liposomal encapsulation of lysosomal enzymes extracted from Saccharomyces cerevisiae. Liposomes were formed with L-α-phosphatidylcholine from egg yolk and cholesterol. To encapsulate whole lysosomal enzymes in liposomes made with and without cholesterol, L-α-phosphatidylcholine and cholesterol were added to chloroform at a ratio of 10:0 (L-α-phosphatidylcholine:cholesterol) and then evaporated for 10 min at 4°C. The residue after evaporation was mixed with lysosomal enzymes at the same ratio and then vortexed for 1 min and sonicated for 5 sec to encapsulate the enzymes. Liposome-encapsulated lysosomal enzymes were created using various amounts of lysosomal enzymes and cholesterol. The results indicated that the optimal encapsulation conditions were lipid:cholesterol ratios of 7:3 and 8:2. Liposome formation was confirmed by TEM imaging. After 1 day, two types of liposomes released small amounts of lysosomal enzymes. However, after 6 days, liposomes formed from mixtures of lipid and cholesterol did not exhibit any changes, whereas liposomes formed from only lipids released high amounts of lysosomal enzymes. Lysosomal enzymes encapsulated in liposomes have potential as important drug delivery carriers, as liposomes are able to control drug release and bioavailability.     

Distribution of negatively charged liposomes on rat liver hepatocytes and non-hepatocytes in cell suspension

The in vitro interactions between negatively charged multilamellar liposomes and purified rat liver parenchymal and non-parenchymal cells were studied. The liposomes were labelled with [¹⁴C]cholesterol and contained [³H]methotrexate. For both cell types the time course of liposomal attachment to the cells slowed down gradually after a rapid initial phase lasting ca 90 min. The rate of attachment at 4 °C was 3–7 times lower than that at 37 °C, and the metabolic inhibitors dinitrophenol and iodoacetic acid caused reduction of 20–30%. Up to 45% of the cell-associated liposomal radioactivity could be detached within 1 h incubation with unlabelled liposomes. Whereas liver parenchymal cell suspension seemed to exhibit similar characteristics in vitro as in vivo, the non-parenchymal cells in vitro showed a 20–50-fold reduction in the rate of liposomal attachment compared to in vivo.