Concentrating dopamine in liposomes using transmembrane gradient of ammonium sulfate]

The active loading of liposomes from egg phosphatidylcholine and cholesterol with dopamine using an ammonium sulfate gradient was studied. Our conditions allowed the enrichment of the monolamellar liposomes with 90% dopamine added to the medium and, thus, considerably improve the dopamine/lipid ratio. Dopamine-containing liposomes with tocopherol in their lipid bilayer were shown to be stable for 3 weeks.     

Supramolecular organization of S12363-liposomes prepared with two different remote loading processes

The S12363 anticancer drug was encapsulated into liposomes in an attempt to increase its therapeutic index. Loading of S12363 was achieved using two different processes based on the formation of either a pH gradient or an ammonium gradient between the acidic inner liposomal compartment and the basic outer phase. High encapsulation yields (> 90%) were obtained using both processes for sphingomyelin/cholesterol/cholesterol-PEG vesicles. Spectrofluorimetry measurements have shown that liposomes were characterized by an internal pH around 4 for both loading processes. This internal pH was stable over a period of at least 20 days. Differential scanning calorimetry coupled with time-resolved synchrotron X-ray diffraction was used to study the drug/carrier supramolecular organization. In ammonium sulfate, S12363 was inserted into the bilayer in the vicinity of the polar headgroup. In citrate buffer, S12363 was mainly adsorbed at the water-lipid interface. The drug partitioning into the membrane was inhomogeneous and led to the formation of drug-rich and drug-poor domains. This effect was enhanced in the presence of cholesterol, especially in ammonium sulfate. To conclude, for both processes, the encapsulated drug was found inside the liposome aqueous core but strongly interacting with the membrane.     

The next generation of liposome delivery systems: recent experience with tumor-targeted,sterically-stabilized immunoliposomes and active-loading gradients

Three topics are discussed. Enhanced anti-tumor efficacy of targeted doxorubicin-containing sterically-stabilized liposomes using an anti-beta1 integrin Fab' ligand. Use of tumor targeting with an internalizing ligand to improve the efficacy of a non-leaky cisplatin-containing sterically-stabilized liposome formulation. Formulation variables (remote-loading with dextran ammonium sulfate, rigid lipid bilayer) used to optimize in vivo performance of a liposomal camptothecin analog.     

Proteins and peptides bound to long-circulating liposomes.

Liposome formulations with prolonged circulation time have recently been developed as a potential sustained-release drug delivery system. Data shown in this report indicate that such formulations can also be used to prolong the circulation time of proteins and peptides by conjugating them to the surface of liposomes. Increase of the circulation halflife ranged from 2- to 150-fold depending on the protein/lipid ratio of the liposomal formulation, liposome size, and the lipid composition of liposomes. Since the proteins/peptides localize on the liposome surface, instead of being entrapped inside the liposomes, they are directly available for binding to its receptor molecules and express the biological activity. This strategy has been successfully applied to two proteins with known fast clearance rate, i.e. asialofetuin and ricin A-chain. The biological activities of both proteins are preserved when they are formulated in liposomes. Incorporation of a peptide, i.e. a-factor of the yeast Saccharomyces cerevisiae, into the liposome membrane also significantly enhanced the circulation time of the peptide.     

Liposomes as carriers of polyfunctional immunogens

Liposomes are promising carriers for construction of the up-to-date chemical vaccinal preparations. The immunogenic and immunomodulating properties of liposomes may be varied by incorporation of the natural or synthetic immunomodulators into the inner water volume or into the lipid bilayer parallel with antigens as well as by introduction of the receptor-specific vector to definite types of immunocompetent cells into liposomes. The pronounced immunobiological properties of the liposomal carrier are shown in studies of liposomes conjugated with haptens or model protein antigens. The application of liposomes as carriers of bacterial antigens induces a delayed catabolism of the antigen and formation of its depot. The immunomodulating properties of antigen-containing liposomes rise after introduction of such immunomodulators as lipid A, muramyldipeptide or interleukin-1 into the liposome composition.     

Differential uptake of liposomes varying in size and lipid composition by parenchymal and kupffer cells of mouse liver

Using liposomes differing in size and lipid composition, we have studied the uptake characteristics of the liver parenchymal and Kupffer cells. Desferal labeled with iron-59 was chosen as a radiomarker for the liposomal content, because Desferal in its free form does not cross cellular membranes. At various time intervals after an intravenous injection of liposomes into mice, the liver was perfused with collagenase, and the cells were separated in a Percoll gradient. It was found that large multilamellar liposomes (diameter of about 0.5 μm) were mainly taken up by the Kupffer cells. For these large liposomes, the rate of uptake by Kupffer cells was rapid, with maximum uptake at around 2 hours after liposome injection. Unexpectedly, small unilamellar liposomes (diameter of about 0.08 μm) were less effectively taken up by Kupffer cells, and the rate of uptake was slow, with a maximum uptake at about 10 hours after liposome injection. In contrast, parenchymal cells were more effective in taking up small liposomes and the uptake of large liposomes was negligible. In addition, liposomes made with a galactolipid as part of the lipid constituents appeared to have higher affinity to parenchymal cells than liposomes made without the galactolipid. These findings should be of importance in designing suitable liposomes for drug targeting.     

Effect of Macrophage Elimination Using Liposome-Encapsulated Dichloromethylene Diphosphonate on Tissue Distribution of Liposomes

Abstract

Effect of macrophage elimination using liposomal dichloromethylene diphosphonate (C1₂MDP)1 on tissue distribution of different types of liposomes was examined in mice. Intravenously administration into mice with CI2MDP encapsulated in liposomes composed of phosphatidylcholine, cholesterol and phosphatidylserine exhibits a temporary blockade of liver and spleen function for liposome uptake. At a low dose of 90 (ig/mouse, the liposome uptake by the liver was significantly decreased. Such decrease was accompanied by an increase in liposome accumulation in either spleen or blood depending on liposome composition and size. Direct correlation between the administration dose of liposomal CI2MDP and the liposome circulation time in blood was also obtained even for liposomes with an average diameter of more than 500 nm. These results indicate that temporary elimination of macrophages of the liver and spleen using liposomal CI2MDP may prove to be useful to enhance the drug delivery efficiency of liposomes.     

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)     

THE NEXT GENERATION OF LIPOSOME DELIVERY SYSTEMS: RECENT EXPERIENCE WITH TUMOR-TARGETED,STERICALLY-STABILIZED IMMUNOLIPOSOMES AND ACTIVE-LOADING GRADIENTS

ABSTRACT

Three topics are discussed. Enhanced anti-tumor efficacy of targeted doxorubicin-containing sterically-stabilized liposomes using an anti-β₁ integrin Fab’ ligand. Use of tumor targeting with an internalizing ligand to improve the efficacy of a non-leaky cisplatin-containing sterically-stabilized liposome formulation. Formulation variables (remote-loading with dextran ammonium sulfate, rigid lipid bilayer) used to optimize in vivo performance of a liposomal camptothecin analog.     

Improved Encapsulation of DNA in pH-Sensitive Liposomes for Transfection

Abstract

A simple method has been developed to prepare liposomes containing large amounts of DNA. The procedure consisted of three cycles of freeze-thawing a mixture of sonicated liposomes and DNA. The encapsulation efficiency depended on the size of DNA. For a small plasmid (2.7 kb), approximately 40% of input DNA was entrapped with an efficiency of 16 μgDNA/μmol lipid. For larger plasmids, the encapsulation efficiency decreased considerably. Transfection of cultured mouse L929 cells mediated by the DNA-containing liposomes was assayed with a plasmid containing the E. coli chloramphenicol acetyl transferase gene. The transfection activity of the liposome was primarily determined by its pH sensitivity. Acid-sensitive liposomes transfected cells efficiently, whereas pH-insensitive liposomes were much less active. The level of the expression of the exogenous gene in the treated cells could be further modulated by protein kinase C (PKC) activators that were incorporated into the liposomal membrane as a minor lipid component. Transfection conditions were optimized with respect to DNA, lipid, and PKC activator concentrations. The results of the current study may help the use of liposomal delivery system for applications in gene therapy.     

The Use of Transmembrane pH Gradient-Driven Drug Encapsulation in the Pharmacodynamic Evaluation of Liposomal Doxorubicin

Abstract

The toxicity and efficacy properties of doxorubicin entrapped inside liposomes are sensitive to the physical characteristics of the vesicle carrier system. Studies addressing such relationships must use preparation procedures with the ability to independently vary vesicle size, lipid composition and drug to lipid ratio while maintaining high trapping efficiencies. The transmembrane pH gradient-driven encapsulation technique allows such liposomal doxorubicin formulations to be prepared. Pharmacokinetic, toxicology and antitumour studies with these systems have revealed several important relationships between liposome physical properties and biological activity. The acute toxicity of liposomal doxorubicin is related primarily to the ability of the liposomes to retain doxorubicin after administration. Including cholesterol and increasing the degree of acyl chain saturation of the phospholipid component in the liposomes significantly decreases drug leakage in the blood, reduces cardiac tissue accumulation of doxorubicin and results in increased LD₅₀ values. In contrast, the efficacy of liposomal doxorubicin is most influenced by liposome size. Specifically, liposomes with a diameter of approximately 100 nm or less exhibit enhanced circulation lifetimes and antitumour activity. While these relationships appear to be rather straightforward, there exist anomalies which suggest that a more thorough evaluation of liposomal doxorubicin pharmacokinetics may be required in order to fully understand its mechanism of action. A key feature in this regard is the ability to differentiate between non-encapsulated and liposome encapsulated doxorubicin pools in the circulation as well as in tumours and normal tissues. This represents a major challenge that must be addressed if significant advances in the design of more effective liposomal doxorubicin formulations are to be achieved.     

Calorimetric study of the interaction of binary DMTAP/DOTAP cationic liposomes with plasmid DNA

Cationic liposomes have been suggested as possible agents for nonviral gene transfer. The interaction of plasmid DNA (pDNA) with dispersions of stable unilamellar cationic liposomes based on the binary lipid system 1,2-dimyristoyl-3-trimethyl-ammonium-propane (DMTAP):1,2-dioleoyl-3-trimethyl-ammonium-propane (DOTAP) has been studied by using isothermal titration calorimetry (ITC), high-precision differential scanning calorimetry (DSC), dynamic light scattering (DLS), and circular dichroism (CD). Systematic calorimetric and DLS exploration of the DMTAP:DOTAP binary system reveals that single-bilayer liposomes are stable at the 4:1 molar ratio, exhibiting the main lipid-phase transition temperature at ~25.3°C, and a total enthalpy change δH = 8.5 ± 0.4 kcal/mol. The interaction of pDNA with unilamellar DMTAP:DOTAP vesicles was investigated by ITC experiments, which clearly distinguished endothermic binding between the phosphate and the ammonium groups from exothermic processes, driven by slow kinetics, corresponding to interliposomal, DNA-triggered aggregation that leads to the formation of large multilamellar liposome/pDNA assemblies. Lipid-added-to-pDNA and pDNA-added-to-lipid experiments have been carried out in order to systematically explore the interaction mechanisms. Complex ITC profiles are revealed, which may be linked to packing rearrangements of the pDNA molecules bound at the outer liposomal surface, possibly due to binding to more than one liposome or due to p-DNA-enhanced heterogeneity in the local lipid concentration. DNA-mediated aggregation effects are detected at high [ammonium]/[phosphate] molar ratios in the case of lipid-added-to-pDNA interactions and at relatively low [phosphate]/[ammonium] molar ratios in the case of pDNA-added-to-lipid.     

Anticancer double lipid prodrugs: liposomal preparation and characterization

The escape of encapsulated anticancer drugs from liposomes by passive diffusion often leads to suboptimal drug concentrations in the cancer tissue, therefore calling for effective trigger mechanisms to release the drug at the target. We investigated mixtures of lipid components that not only form stable liposomes, but also can be turned into active drugs by secretory phospholipase A? (sPLA?), an enzyme that is upregulated in various cancer cells, without the necessity for conventional liposome drug loading. The liposomes are composed of a novel lipid-based retinoid prodrug premixed with saturated phospholipids. The prodrug is found to be miscible with phospholipids, and the lipid mixtures are shown to form liposomes with the desired size distribution. The preparation procedure, phase behavior, and physicochemical properties of the formed liposomes are described as a function of lipid composition. We show that the premixing of the prodrug with phospholipids can be used to modify the physicochemical properties of liposomal formulations. The results should prove useful for further exploration of the potential for using these novel lipid prodrugs in liposomal formulations for cancer treatment.     

Design and characterization of enzymosomes with surface-exposed superoxide dismutase

Superoxide dismutase (SOD) was chemically modified by covalent linkage of fatty acid chains to the accessible ε-amino groups of the enzyme. This acylation method gave rise to a different enzyme entity (Ac-SOD) as evidenced by different physicochemical properties such as octanol/water partition coefficient and isoelectric point (pI) as compared to SOD. Ac-SOD was incorporated in conventional and long-circulating liposomes (LCL) and characterized in terms of incorporation efficiency, protein to lipid ratio (Prot/Lip), enzymatic activity retention and zeta potential. The observation that Ac-SOD liposomes present enzymatic activity on their external surface indicates that these formulations can act independent of rate and extent of enzyme release as required in case of SOD liposomes. The decrease of superficial charge of liposomal formulations containing Ac-SOD, as compared to SOD liposomes, may be related to the negatively charged enzyme molecules localized on the liposome surface. The comparative characterization of Ac-SOD and SOD liposomal formulations evidenced that the two enzyme forms differ substantially regarding their intraliposomal location: SOD tends to be localized in the internal aqueous spaces, whereas Ac-SOD is expected to be localized in the lipid bilayers of the liposomes, partially buried into the outer surface and exposed to the external medium. These liposomal structures with surface-exposed SOD were designated as Ac-SOD enzymosomes. The properties of these enzymosomes may influence the therapeutic effect, as the release of the enzyme from extravasated vesicles is no longer a necessary requirement for achieving dismutating activity within the inflamed target site.     

Drug release rate influences the pharmacokinetics, biodistribution, therapeutic activity, and toxicity of pegylated liposomal doxorubicin formulations in murine breast cancer

The pharmacokinetics (PK), biodistribution (BD), and therapeutic activity of pegylated liposomal doxorubicin formulations with different drug release rates were studied in an orthotopic 4T1 murine mammary carcinoma model. The focus of these experiments was to study the effects of different release rates on the accumulation of liposomal lipid and doxorubicin (DXR) into the tumor and cutaneous tissues of mice (skin and paws). These tissues were chosen because the clinical formulation of pegylated liposomal doxorubicin (Caelyx)/Doxi) causes mucocutaneous reactions such as palmar-plantar erythrodysesthesia (PPE). Liposomes with different doxorubicin (DXR) leakage rates were prepared by altering liposome fluidity through changing the fatty acyl chain length and/or degree of saturation of the phosphatidylcholine component of the liposome. Liposomes with fast, intermediate, and slow rates of drug release were studied. The plasma PK of the liposomal lipid was similar for all formulations, while the plasma PK of the DXR component was dependent on the liposome formulation. Liposomal lipid accumulated to similar levels in tumor and cutaneous tissues for all three formulations tested, while the liposomes with the slowest rates of DXR release produced the highest DXR concentrations in both cutaneous tissues and in tumor. Liposomes with the fastest drug release rates resulted in low DXR concentrations in cutaneous tissues and tumor. The formulation with intermediate release rates produced unexpected toxicity that was not related to the lipid content of the formulation. The liposomes with the slowest rate of drug leakage had the best therapeutic activity of the formulations tested.     

Tissue distribution of EDTA encapsulated within liposomes of varying surface properties

Liposomes containing ethylenediaminetetraacetic acid (EDTA) were prepared with different surface properties by varying the liposomal lipid constituents. Positively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and stearylamine. Negatively charged liposomes were prepared with a mixture of phosphatidylcholine, cholesterol, and phosphatidylserine. Neutral liposomes were prepared with phosphatidylcholine alone, dipalmitoyl phosphatidylcholine alone, or with a mixture of phosphatidylcholine and cholesterol. Distributions of ¹⁴C-labeled EDTA were determined in mouse tissues from 5 min to 24 h after a single intravenous injection of liposome preparation. Differences in tissue distribution were produced by the different liposomal lipid compositions. Uptake of EDTA by spleen and marrow was highest from negatively charged liposomes. Uptake of EDTA by lungs was highest from positively charged liposomes; lungs and brain retained relatively high levels of EDTA from these liposomes between 1 and 6 h after injection. Liver uptake of EDTA from positively or negatively charged liposomes was similar; the highest EDTA uptake by liver was from the neutral liposomes composed of a mixture of phosphatidylcholine and cholesterol. Liposomes composed of dipalmitoyl phosphatidylcholine produced the lowest liposomal EDTA uptake observed in liver and marrow but modrate uptake by lungs. Tissue uptake and retention of EDTA from all of the liposome preparations were greater than those of non-encapsulated EDTA. The results presented demonstrate that the tissue distribution of a molecule can be modified by encapsulation of that substance into liposomes of different surface properties. Selective delivery of liposome-encapsulated drugs to specific tissues could be effectively used in chemotherapy and membrane biochemistry.     

Oral delivery of antigens in liposomes with some lipid compositions modulates oral tolerance to the antigens

Several liposomes containing ovalbumin (OVA), a model antigen, with different lipid compositions were prepared in order to evaluate their ability to induce oral tolerance. Oral administration of these liposomal OVAs induced suppression of the proliferative responses of popliteal lymph node cells from the treated mice to OVA, suggesting that these treated mice were tolerized. The efficiency of the induction of oral tolerance was affected by the liposome composition. OVA entrapment in these liposomes could modulate the tolerizing dose of OVA itself. These results suggest that some liposomes can be suitable antigen-delivery systems for modulated and/or effective induction of oral tolerance.     

Evaluation and optimization of DNA delivery into gliosarcoma 9L cells by a cholesterol-based cationic liposome

This paper reports results concerning the transfection of gliosarcoma cells 9L using an original cholesterol-based cationic liposome as carrier. This cationic liposome was prepared from triethyl aminopropane carbamoyl cholesterol (TEAPC-Chol) and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE). The used concentration of liposome was not cytotoxic as revealed by the MTT test. TEAPC-Chol/DOPE liposomes allowed the plasmids encoding reporter genes to enter the nucleus as observed both by electron microscopy and functionality tests using fluorescence detection of green fluorescent protein (GFP) and luminometric measurements of luciferase activity. By changing the cationic lipid/DNA molar charge ratio, optimal conditions were determined. Further, improvement of the transfection level has been obtained by either precondensing plasmid DNA with poly-l-lysine or by adding polyethylene glycol (PEG) in the transfection medium. The optimal conditions determined are different depending on whether the transfection is made with cells in culture or with tumors induced by subcutaneous (s.c.) injection of cells in Nude mice. For in vivo assays, a simple method to overcome the interference of haemoglobin with the chemiluminescence intensity of luciferase has been used. These results would be useful for gaining knowledge about the potential for the cationic liposome TEAPC-Chol/DOPE to transfect brain tumors efficiently.     

Evaluation and optimization of DNA delivery into gliosarcoma 9L cells by a cholesterol-based cationic liposome

This paper reports results concerning the transfection of gliosarcoma cells 9L using an original cholesterol-based cationic liposome as carrier. This cationic liposome was prepared from triethyl aminopropane carbamoyl cholesterol (TEAPC-Chol) and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE). The used concentration of liposome was not cytotoxic as revealed by the MTT test. TEAPC-Chol/DOPE liposomes allowed the plasmids encoding reporter genes to enter the nucleus as observed both by electron microscopy and functionality tests using fluorescence detection of green fluorescent protein (GFP) and luminometric measurements of luciferase activity. By changing the cationic lipid/DNA molar charge ratio, optimal conditions were determined. Further, improvement of the transfection level has been obtained by either precondensing plasmid DNA with poly-L-lysine or by adding polyethylene glycol (PEG) in the transfection medium. The optimal conditions determined are different depending on whether the transfection is made with cells in culture or with tumors induced by subcutaneous (s.c.) injection of cells in Nude mice. For in vivo assays, a simple method to overcome the interference of haemoglobin with the chemiluminescence intensity of luciferase has been used. These results would be useful for gaining knowledge about the potential for the cationic liposome TEAPC-Chol/DOPE to transfect brain tumors efficiently.     

pH gradient loading of anthracyclines into cholesterol-free liposomes: enhancing drug loading rates through use of ethanol

Application of cholesterol-free liposomes as carriers for anticancer drugs is hampered, in part, because of standard pH gradient based loading methods that rely on incubation temperatures above the phase transition temperature (Tc) of the bulk phospholipid to promote drug loading. In the absence of cholesterol, liposome permeability is enhanced at these temperatures which, in turn, can result in the collapse of the pH gradient and/or unstable loading. Doxorubicin loading studies, for example, indicate that the drug could not be loaded efficiently into cholesterol-free DSPC liposomes. We demonstrated that this problem could be circumvented by the addition of ethanol as a permeability enhancer. Doxorubicin loading rates in cholesterol-free DSPC liposomes were 6.6-fold higher in the presence of ethanol. In addition, greater than 90% of the added doxorubicin was encapsulated within 2 h at 37 degrees C, an efficiency that was 2.3-fold greater than that observed in the absence of ethanol. Optimal ethanol concentrations ranged from 10% to 15% (v/v) and these concentrations did not significantly affect liposome size, retention of an aqueous trap marker (lactose) or, most importantly, the stability of the imposed pH gradient. Cryo-transmission electron micrographs of liposomes exposed to increasing concentrations of ethanol indicated that at 30% (v/v) perturbations to the lipid bilayer were present as evidenced by the appearance of open liposomes and bilayer sheets. Ethanol-induced increased drug loading was temperature-, lipid composition- and lipid concentration-dependent. Collectively, these results suggest that ethanol addition to preformed liposomes is an effective method to achieve efficient pH gradient-dependent loading of cholesterol-free liposomes at temperatures below the Tc of the bulk phospholipid.