Formation of transition metal-doxorubicin complexes inside liposomes

Doxorubicin complexation with the transition metal manganese (Mn²⁺) has been characterized, differentiating between the formation of a doxorubicin-metal complex and doxorubicin fibrous-bundle aggregates typically generated following ion gradient-based loading procedures that rely on liposome encapsulated citrate or sulfate salts. The physical and chemical characteristics of the encapsulated drug were assessed using cryo-electron microscopy, circular dichroism (CD) and absorbance spectrophotometric analysis. In addition, in vitro and in vivo drug loading and release characteristics of the liposomal formulations were investigated. Finally, the internal pH after drug loading was measured with the aim of linking formation of the Mn²⁺ complex to the presence or absence of a transmembrane pH gradient. Doxorubicin was encapsulated into either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/cholesterol (Chol) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/Chol liposomes, where the entrapped salts were citrate, MnSO₄ or MnCl₂. In response to a pH gradient or a Mn²⁺ ion gradient, doxorubicin accumulated inside to achieve a drug-to-lipid ratio of approximately 0.2:1 (wt/wt). Absorbance and CD spectra of doxorubicin in the presence of Mn²⁺ suggested that there are two distinct structures captured within the liposomes. In the absence of added ionophore A23187, drug loading is initiated on the basis of an established pH gradient; however, efficient drug uptake is not dependent on maintenance of the pH gradient. Drug release from DMPC/Chol is comparable regardless of whether doxorubicin is entrapped as a citrate-based aggregate or a Mn²⁺ complex. However, in vivo drug release from DSPC/Chol liposomes indicate less than 5% or greater than 50% drug loss over a 24-h time course when the drug was encapsulated as an aggregate or a Mn²⁺ complex, respectively. These studies define a method for entrapping drugs possessing coordination sites capable of complexing transition metals and suggest that drug release is dependent on lipid composition, internal pH, as well as the nature of the crystalline precipitate, which forms following encapsulation.     

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.     

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 °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.     

Arsenic trioxide liposomes: encapsulation efficiency and in vitro stability

The use of arsenic-containing compounds in cancer therapy is currently being re-considered, after the recent approval of arsenic trioxide (Trisenox) for the treatment of relapsed promyelocytic leukemia (PML). In an attempt to prepare a carrier system to minimize the toxicity of this drug, the aim of this study is to prepare and characterize liposomes encapsulating arsenic trioxide (ATO). For this, we prepared different types of liposomes entrapping ATO: large multilamellar (MLV), sonicated (SUV) and dried reconstituted vesicles (DRV). The techniques used were: thin film hydration, sonication and the DRV method, respectively. Two lipid compositions were studied for each liposome type, EggPC/Chol (1:1) and DSPC/Chol (1:1). After liposome preparation, drug encapsulation was evaluated by measuring arsenic in liposomes. For this, energy-dispersive X-ray fluorescence spectroscopy or atomic absorption was used. In addition, the retention of the drug in the liposomes was evaluated after incubating the liposomes in buffer at 37 degrees C. The experimental results reveal that encapsulation of ATO in liposomes ranges between 0.003 and 0.506 mol/ mol of lipid, and is highest in the DRV vesicles and lowest in the small unilamellar vesicles, as anticipated. Considering the in vitro stability of ATO-encapsulating liposomes: 1) For the PC/Chol liposomes (DRV and MLV), after 24 hours of incubation, more than 70% (or 90% in some cases) of the initially encapsulated amount of ATO was released. 2) The liposomes composed of DSPC/Chol could retain substantially higher amounts of ATO, especially the DRV liposomes (54% retained after 24 h). 3) In the case of PC/Chol, temperature of incubation has no effect on the ATO release after 24 hours, but affects the rate of ATO release in the MLV liposomes, while for the DSPC/Chol liposomes there is a slight increase (statistically insignificant) of ATO release at higher temperature.     

Arsenic Trioxide Liposomes: Encapsulation Efficiency and In Vitro Stability

The use of arsenic‐containing compounds in cancer therapy is currently being re‐considered, after the recent approval of arsenic trioxide (Trisenox®) for the treatment of relapsed promyelocytic leukemia (PML). In an attempt to prepare a carrier system to minimize the toxicity of this drug, the aim of this study is to prepare and characterize liposomes encapsulating arsenic trioxide (ATO). For this, we prepared different types of liposomes entrapping ATO: large multilamellar (MLV), sonicated (SUV) and dried reconstituted vesicles (DRV). The techniques used were: thin film hydration, sonication and the DRV method, respectively. Two lipid compositions were studied for each liposome type, EggPC/Chol (1:1) and DSPC/Chol (1:1). After liposome preparation, drug encapsulation was evaluated by measuring arsenic in liposomes. For this, energy‐dispersive X‐ray fluorescence spectroscopy or atomic absorption was used. In addition, the retention of the drug in the liposomes was evaluated after incubating the liposomes in buffer at 37°C. The experimental results reveal that encapsulation of ATO in liposomes ranges between 0.003 and 0.506 mol/ mol of lipid, and is highest in the DRV vesicles and lowest in the small unilamellar vesicles, as anticipated. Considering the in vitro stability of ATO‐encapsulating liposomes: 1) For the PC/Chol liposomes (DRV and MLV), after 24 hours of incubation, more than 70% (or 90% in some cases) of the initially encapsulated amount of ATO was released. 2) The liposomes composed of DSPC/Chol could retain substantially higher amounts of ATO, especially the DRV liposomes (54% retained after 24 h). 3) In the case of PC/Chol, temperature of incubation has no effect on the ATO release after 24 hours, but affects the rate of ATO release in the MLV liposomes, while for the DSPC/Chol liposomes there is a slight increase (statistically insignificant) of ATO release at higher temperature.     

Characterization of liposomal systems containing doxorubicin entrapped in response to pH gradients

Studies from this laboratory (Mayer et al. (1986) Biochim. Biophys. Acta 857, 123-126) have shown that doxorubicin can be accumulated into liposomal systems in response to transmembrane pH gradients (inside acidic). Here, detailed characterizations of the drug uptake and retention properties of these systems are performed. It is shown that for egg phosphatidylcholine (EPC) vesicles (mean diameter of 170 nm) exhibiting transmembrane pH gradients (inside acidic) doxorubicin can be sequestered into the interior aqueous compartment to achieve drug trapping efficiencies in excess of 98% and drug-to-lipid ratios of 0.36:1 (mol/mol). Drug-to-lipid ratios as high as 1.7:1 (mol/mol) can be obtained under appropriate conditions. Lower drug-to-lipid ratios are required to achieve trapping efficiencies in excess of 98% for smaller (less than or equal to 100 nm) systems. Doxorubicin trapping efficiencies and uptake capacities are related ito maintenance of the transmembrane pH gradient during encapsulation as well as the interaction between doxorubicin and entrapped citrate. This citrate-doxorubicin interaction increases drug uptake levels above those predicted by the Henderson-Hasselbach relationship. Increased drug-to-lipid ratios and trapping efficiencies are observed for higher interior buffering capacities. Retention of a large transmembrane pH gradient (greater than 2 units) after entrapment reduces the rate of drug leakage from the liposomes. For example, EPC/cholesterol (55:45, mol/mol) liposomal doxorubicin systems can be achieved which released less than 5% of encapsulated doxorubicin (drug-to-lipid molar ratio = 0.33:1) over 24 h at 37 degrees C. This pH gradient-dependent encapsulation technique is extremely versatile, and well characterized liposomal doxorubicin preparations can be generated to exhibit a wide range of properties such as vesicle size, lipid composition, drug-to-lipid ratio and drug release kinetics. This entrapment procedure therefore appears well suited for use in therapeutic applications. Finally, a rapid colorimetric test for determining the amount of unencapsulated doxorubicin in liposomal systems is described.     

Doxorubicin Encapsulated in Long-Circulating Thermosensitive Liposomes

Abstract

Doxorubicin (DXR) was encapsulated in long-circulating, thermosensitive liposomes (TSL, 180-200 nm in mean diameter), prepared from dipalmitoyl phosphatidyl choline (DPPC)/distearoyl phosphatidyl choline (DSPC) (9:1, m/m) and either 3 mol% of amphipathic polyethylene glycol (PEG) with 1000 in average molecular weight or 6 mol% of ganglioside GMI (GMI), with 95-98% entrapping efficiency by the pH gradient method. 57% or 45% of the entrapped DXR was released from PEG/DPPC/DSPC or G_M1/DPPC/DSPC liposomes, respectively, by incubation with 20% serum at 42°C for 5 min. Inclusion of PEG or G_M1 endowed TSL with prolonged circulation ability, resulting in increased blood levels of liposomes and decreased reticuloendothelial system (RES) uptake over 6 hours after injection. Concomitantly, high DXR level in blood was kept for long time.

Accumulation of DXR into tumor tissue of tumor-bearing mice (mouse colon carcinoma 26) by local hyperthermia after injection of DXR-long-circulating TSL was 2 times or 7 times higher than that after treatment with DXR-TSL liposomes or free DXR in combination with hyperthermia, respectively. Furthermore, the systemic treatment with DXR-long-circulating TSL and hyperthermia resulted in effective tumor growth retardation and increased survival time. These results indicate that the combination of long-circulating, thermosensitive liposomes with local hyperthermia at the tumor site could be clinically useful for delivering a wide range of chemotherapeutic agents in the treatment of solid tumors.     

Remote loading of doxorubicin into liposomes driven by a transmembrane phosphate gradient

This study examines a new method for the remote loading of doxorubicin into liposomes. It was shown that doxorubicin can be loaded to a level of up to 98% into large unilamellar vesicles composed of egg phosphatidylcholine/cholesterol (7/3 mol/mol) with a transmembrane phosphate gradient. The different encapsulation efficiencies which were achieved with ammonium salts (citrate 100%, phosphate 98%, sulfate 95%, acetate 77%) were significantly higher as compared to the loading via sodium salts (citrate 54%, phosphate 52%, sulfate 44%, acetate 16%). Various factors, including pH-value, buffer capacity, solubility of doxorubicin in different salt solutions and base counter-flow, which likely has an influence on drug accumulation in the intraliposomal interior are taken into account. In contrast to other methods, the newly developed remote loading method exhibits a pH-dependent drug release property which may be effective in tumor tissues. At physiological pH-value doxorubicin is retained in the liposomes, whereas drug release is achieved by lowering the pH to 5.5 (approximately 25% release at 25 degrees C or 30% at 37 degrees C within two h). The DXR release of liposomes which were loaded via a sulfate gradient showed a maximum of 3% at pH 5.5.     

Remote loading of doxorubicin into liposomes driven by a transmembrane phosphate gradient

This study examines a new method for the remote loading of doxorubicin into liposomes. It was shown that doxorubicin can be loaded to a level of up to 98% into large unilamellar vesicles composed of egg phosphatidylcholine/cholesterol (7/3 mol/mol) with a transmembrane phosphate gradient. The different encapsulation efficiencies which were achieved with ammonium salts (citrate 100%, phosphate 98%, sulfate 95%, acetate 77%) were significantly higher as compared to the loading via sodium salts (citrate 54%, phosphate 52%, sulfate 44%, acetate 16%). Various factors, including pH-value, buffer capacity, solubility of doxorubicin in different salt solutions and base counter-flow, which likely has an influence on drug accumulation in the intraliposomal interior are taken into account. In contrast to other methods, the newly developed remote loading method exhibits a pH-dependant drug release property which may be effective in tumor tissues. At physiological pH-value doxorubicin is retained in the liposomes, whereas drug release is achieved by lowering the pH to 5.5 (approximately 25% release at 25 °C or 30% at 37 °C within two h). The DXR release of liposomes which were loaded via a sulfate gradient showed a maximum of 3% at pH 5.5.     

Liposomal Anticancer Drugs as Agents to be used in Combination with other Anticancer Agents: Studies on a Liposomal Formulation with two Encapsulated Drugs

Abstract

When considering the use of combination therapies with liposomal anticancer agents several approaches can be defined. One approach could rely on administration of one liposomal formulation with more than one entrapped cytotoxic drug. This study focuses on an assessment of a liposomal formulation containing vincristine and mitoxantrone. Distearoyl phosphatidylcholine (DSPC)/Cholesterol (Choi) (55:45 molar ratio) liposomes were loaded with vincristine using transmembrane pH gradients. These systems were subsequently incubated with mitoxantrone to effect uptake of the second drug. Retention of both drugs was determined in vitro and in vivo. In vitro drug release indicated >95% retention of mitoxantrone and approximately 75% retention of vincristine when liposomes were prepared with an initial interior pH of 2.0. In vivo results however, demonstrated that greater than 80% of the encapsulated vincristine was released within 1 hour following i.v. administration. The instability of a liposomal formulation containing two anticancer drugs following i.v. administration may be a consequence of a combination of factors including drug-loading induced collapse of the transmembrane pH gradient, loss due to osmotic effects and an associated insertion of serum proteins into the bilayer, as well as the presence of a large biological “sink” which can alter the transbilayer drug gradient in favor of drug release.     

Optimization of Drug Loading Procedures and Characterization of Liposomal Formulations of Two Novel Agents Intended for Boron Neutron Capture Therapy (BNCT)

Abstract

The characterization of two liposomal formulations of boronated DNA-interacting agents has been performed. It is shown that the two boronated drugs, WSA-Water Soluble Acridine and WSP-Water Soluble Phenantridine, can be encapsulated within unilamellar sterically stabilized liposomes with high drug-to-lipid ratios (up to 0.50:1 (mol:mol)), using transmembrane pH gradients. The steric stabilization of the liposomes was accomplished by the addition of DSPE-PEG(2000) (PEG-lipid) to DSPC/Cho lipid mixtures and the composition used was DSPC: Cho: DSPE-PEG 55:40:5 (moI%). The loading of the drugs resulted in drug precipitation in the liposomal aqueous core as observed by cryo-transmission electron microscopy (c-TEM). Moreover, it is shown that when pH gradients across the bilayer were used for remote loading of WSP or when ammonium sulfate gradients were used for remote loading of WSA, the formation of small bilayer fragments (discs) was induced. We present compelling evidence that the formation of discs is a consequence of precipitate growth in the liposomal interior. The precipitate growth causes some of the liposomes to rupture resulting in the above mentioned disc-formation and a substantial decrease in trapping efficiency. The in vitro stability of the drug loaded liposomes was excellent, both in buffer and in 25% human serum. For most of the formulations, the release of the drugs was below or around 10% after 24 hours at 37^oC. Furthermore, the influence of initial internal pH and internal buffering capacity on release properties of WSA and WSP were investigated. It is shown that the release profiles of the drugs can be controlled, to a large extent, by varying the composition of the internal liposomal aqueous phase.     

Membrane permeabilizing activity of amphotericin B is affected by chain length of phosphatidylcholine added as minor constituent

The effect of acyl-chain length of phospholipid on the membrane permeabilizing activity of amphotericin B (AmB) was examined using egg phosphatidylcholine (eggPC) liposomes containing 5% or 20% phosphatidylcholine with various lengths of fatty acyl chains from C(10) to C(18); 1,2-dicapryloyl-sn-glycero-3-phosphocholine (DCPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The membrane activity of AmB was evaluated by two methods; the drug was added to a liposome suspension (added-via-aqua), or mixed with lipids prior to liposome preparation (mixed-with-lipid). In both cases, K(+) influx by AmB was measured as pH change inside liposomes by 31P-NMR. The C(10) and C(12) acyl phospholipids markedly enhanced the activity of AmB, the C(14) and C(16) lipids virtually showed no effect, and the C(18) lipid was inhibitory to the AmB's action. Clear distinction between the C(12) and C(14) lipids, which differ only in acyl chains by two carbons, implies that molecular interaction between phospholipid and AmB is partly due to the matching of their hydrophobic length.     

Enhanced Delivery and Antitumor Effect of Doxorubicin Encapsulated in Long-Circulating Liposomes

Abstract

Long-circulating liposomes containing amphipathic polyethyleneglycol (PEG) or ganglioside GM1 (GM1) have been tested for their utility as enhanced delivery system of doxorubicin (DXR) in vivo. DXR was entrapped into liposomes by pH gradient method.

The long-circulating LUV (200 nm in size) composed of DSPC/CH (1:1, m/m) and either 6 mol% of DSPE-PEG1000 or GM1 entrapped DXR with >95% in trapping efficiency. DXR-long-circulating LUVs were administered to leukemic (LI210) mice via the tail vein at a dose of 5mg DXR/kg. The high blood concentration was kept for long time, and significantly increased survival time was observed as compared with free DXR and DXR-LUV. The data indicated that DXR was slowly released from long-circulating LUV during that stayed in bloodstream for long time. Administration of DXR-long-circulating SUV (100 nm) to the colon 26 bearing mice produced the increased DXR level in tumor compared with bear SUV or free drug did, respectively, and resulted in effective tumor growth retardation and increased survival time. DXR was delivered to tumor by accumulation of SUVs themselves.

Long-circulating thermosensitive liposomes (TSL) were prepared from DPPC /DSPC (9:1, m/m) and 3-6 mol% of PEG1000 or GM1. DXR was entrapped with >95% in trapping efficiency. Accumulation of DXR into tumor tissue by local hyperthermia after injection of DXR-long-circulating TSL to colon 26 bearing mice was significantly higher man that of DXR-bare TSL or free DXR, and resulted in effective tumor growth retardation and increased survival time. It was suggested that the entrapped DXR was efficiently released from long-circulating TSL by hyperthermia at the tumor site and entered the tumor tissue by simple diffusion.     

Fluorometric determination in biological fluids of the release kinetics of liposome-entrapped doxorubicin

Abstract

A fluorometric method is presented that allows a continuous monitoring in 50% (v/v) human serum of the release of liposome-entrapped doxorubicin (DXR).This method exploits the intrinsic fluorescence of DXR and uses DNA as a fluorescence quencher of the released drug. It is much more simple and rapid than the commonly used methods. It is shown that this method can be applied to the study of the stability of liposomal-DXR encapsulated using either an ammonium sulfate gradient or a pH gradient. Small unilamellar vesicles (SUVs) made from egg yolk phosphatidylcholines (EPC) were much less stable, either in phosphate buffer saline or in human serum, than SUVs made from a 1/1 mixture of EPC and cholesterol (Choi). SUVs made from EPC/Chol loaded with DXR using an ammonium sulfate gradient were found to be more stable than those loaded using a pH gradient.     

Coencapsulation of irinotecan and floxuridine into low cholesterol-containing liposomes that coordinate drug release in vivo

A liposomal delivery system that coordinates the release of irinotecan and floxuridine in vivo has been developed. The encapsulation of floxuridine was achieved through passive entrapment while irinotecan was actively loaded using a novel copper gluconate/triethanolamine based procedure. Coordinating the release rates of both drugs was achieved by altering the cholesterol content of distearoylphosphatidylcholine (DSPC)/distearoylphosphatidylglycerol (DSPG) based formulations. The liposomal retention of floxuridine in plasma after intravenous injection was dramatically improved by decreasing the cholesterol content of the formulation below 20 mol%. In the case of irinotecan, the opposite trend was observed where increasing cholesterol content enhanced drug retention. Liposomes composed of DSPC/DSPG/Chol (7:2:1, mole ratio) containing co-encapsulated irinotecan and floxuridine at a 1:1 molar ratio exhibited matched leakage rates for the two agents so that the 1:1 ratio was maintained after intravenous administration to mice. The encapsulation of irinotecan was optimal when copper gluconate/triethanolamine (pH 7.4) was used as the intraliposomal buffer. The efficiency of irinotecan loading was approximately 80% with a starting drug to lipid molar ratio of 0.1/1. Leakage of floxuridine from the liposomes during irinotecan loading at 50 °C complicated the ability to readily achieve the target 1:1 irinotecan/floxuridine ratio inside the formulation. As a result, a procedure for the simultaneous encapsulation of irinotecan and floxuridine was developed. This co-encapsulation method has the advantage over sequential loading in that extrusion can be performed in the absence of chemotherapeutic agents and the drug/drug ratios in the final formulation can be more precisely controlled.     

Targeted delivery and triggered release of liposomal doxorubicin enhances cytotoxicity against human B lymphoma cells.

Dioleoylphosphatidylethanolamine (DOPE)-containing liposomes that demonstrated pH-dependent release of their contents were stabilized in the bilayer form through the addition of a cleavable lipid derivative of polyethylene glycol (PEG) in which the PEG was attached to a lipid anchor via a disulfide linkage (mPEG-S-S-DSPE). Liposomes stabilized with either a non-cleavable PEG (mPEG-DSPE) or mPEG-S-S-DSPE retained an encapsulated dye at pH 5.5, but treatment at pH 5.5 of liposomes stabilized with mPEG-S-S-DSPE with either dithiothreitol or cell-free extracts caused contents release due to cleavage of the PEG chains and concomitant destabilization of the DOPE liposomes. While formulations loaded with doxorubicin (DXR) were stable in culture media, DXR was rapidly released in human plasma. pH-Sensitive liposomes, targeted to the CD19 epitope on B-lymphoma cells, showed enhanced DXR delivery into the nuclei of the target cells and increased cytotoxicity compared to non-pH-sensitive liposomes. Pharmacokinetic studies suggested that mPEG-S-S-DSPE was rapidly cleaved in circulation. In a murine model of B-cell lymphoma, the therapeutic efficacy of an anti-CD19-targeted pH-sensitive formulation was superior to that of a stable long-circulating formulation of targeted liposomes despite the more rapid drug release and clearance of the pH-sensitive formulation. These results suggest that targeted pH-sensitive formulations of drugs may be able to increase the therapeutic efficacy of entrapped drugs.     

Coencapsulation of irinotecan and floxuridine into low cholesterol-containing liposomes that coordinate drug release in vivo

A liposomal delivery system that coordinates the release of irinotecan and floxuridine in vivo has been developed. The encapsulation of floxuridine was achieved through passive entrapment while irinotecan was actively loaded using a novel copper gluconate/triethanolamine based procedure. Coordinating the release rates of both drugs was achieved by altering the cholesterol content of distearoylphosphatidylcholine (DSPC)/distearoylphosphatidylglycerol (DSPG) based formulations. The liposomal retention of floxuridine in plasma after intravenous injection was dramatically improved by decreasing the cholesterol content of the formulation below 20 mol%. In the case of irinotecan, the opposite trend was observed where increasing cholesterol content enhanced drug retention. Liposomes composed of DSPC/DSPG/Chol (7:2:1, mole ratio) containing co-encapsulated irinotecan and floxuridine at a 1:1 molar ratio exhibited matched leakage rates for the two agents so that the 1:1 ratio was maintained after intravenous administration to mice. The encapsulation of irinotecan was optimal when copper gluconate/triethanolamine (pH 7.4) was used as the intraliposomal buffer. The efficiency of irinotecan loading was approximately 80% with a starting drug to lipid molar ratio of 0.1/1. Leakage of floxuridine from the liposomes during irinotecan loading at 50 degrees C complicated the ability to readily achieve the target 1:1 irinotecan/floxuridine ratio inside the formulation. As a result, a procedure for the simultaneous encapsulation of irinotecan and floxuridine was developed. This co-encapsulation method has the advantage over sequential loading in that extrusion can be performed in the absence of chemotherapeutic agents and the drug/drug ratios in the final formulation can be more precisely controlled.     

Improved retention of idarubicin after intravenous injection obtained for cholesterol-free liposomes

To date there has been a focus on the application of sterically stabilized liposomes, composed of saturated diacylphospholipid, polyethylene glycol (PEG) conjugated lipids (5-10 mole%) and cholesterol (CH) (>30 mole%), for the systemic delivery of drugs. However, we are now exploring the utility of liposome formulations composed of diacylphospholipid conjugated PEG mixtures prepared in the absence of added cholesterol, with the primary objective of developing formulations that retain encapsulated drug better than comparable formulations prepared with cholesterol. In this report the stability of cholesterol-free distearoylphosphatidylcholine (DSPC):distearoylphosphatidylethanolamine (DSPE)-PEG(2000) (95:5 mol/mol) liposomes was characterized in comparison to cholesterol-containing formulations DSPC:CH (55:45 mol/mol) and DSPC:CH:DSPE-PEG(2000) (50:45:5 mol/mol/mol), in vivo. Circulation longevity of these formulations was determined in consideration of variables that included varying phospholipid acyl chain length, PEG content and molecular weight. The application of cholesterol-free liposomes as carriers for the hydrophobic anthracycline antibiotic, idarubicin (IDA), was assessed. IDA was encapsulated using a transmembrane pH gradient driven process. To determine stability in vivo, pharmacokinetic studies were performed using 'empty' and drug-loaded [(3)H]cholesteryl hexadecyl ether radiolabeled liposomes administered intravenously to Balb/c mice. Inclusion of 5 mole% of DSPE-PEG(2000) or 45 mole% cholesterol to DSPC liposomes increased the mean plasma area under the curve (AUC(0-24h)) 19-fold and 10-fold, respectively. Cryo-transmission electron micrographs of IDA loaded liposomes indicated that the drug formed a precipitate within liposomes. The mean AUC(0-4h) for free IDA was 0.030 micromole h/ml as compared to 1.38 micromole h/ml determined for the DSPC:DSPE-PEG(2000) formulation, a 45-fold increase, demonstrating that IDA was retained better in cholesterol-free compared to cholesterol-containing liposomes.     

Carboxylated phytosterol derivative-introduced liposomes for skin environment-responsive transdermal drug delivery system

Abstract

Transdermal drug delivery systems are a key technology for skin-related diseases and for cosmetics development. The delivery of active ingredients to an appropriate site or target cells can greatly improve the efficacy of medical and cosmetic agents. For this study, liposome-based transdermal delivery systems were developed using pH-responsive phytosterol derivatives as liposome components. Succinylated phytosterol (Suc-PS) and 2-carboxy-cyclohexane-1-carboxylated phytosterol (CHex-PS) were synthesized by esterification of hydroxy groups of phytosterol. Modification of phytosterol derivatives on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes was confirmed by negatively zeta potentials at alkaline pH and the change of zeta potentials with decreasing pH. In response to acidic pH and temperatures higher than body temperature, Suc-PS-containing and CHex-PS-containing liposomes exhibited content release at intracellular acidic compartments of the melanocytes at the basement membrane of the skin. Phytosterol-derivative-containing liposomes were taken up by murine melanoma-derived B16-F10 cells. These liposomes delivered their contents into endosomes and cytosol of B16-F10 cells. Furthermore, phytosterol-derivative-containing liposomes penetrated the 3?D skin models and reached the basement membrane. Results show that pH-responsive phytosterol-derivative-containing DMPC liposomes are promising for use in transdermal medical or cosmetic agent delivery to melanocytes.     

Triggered release of doxorubicin following mixing of cationic and anionic liposomes

In many applications, an ability of liposomes to retain drug and then rapidly release it at some later time would be of benefit. In this work, we investigate the ability of cationic large unilamellar vesicles (LUV) to promote rapid release of doxorubicin from anionic LUV. It is shown that the addition of cationic liposomes containing cholesterol, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) and the cationic lipid N,N-dioleyl-N,N-dimethylammonium chloride (DODAC) to doxorubicin-containing LUV composed of cholesterol, DOPE, DSPC and the anionic lipid dioleoyphosphatidylglycerol (DOPG) can result in release of more than 90% of the drug in times of 30 s or less. Further, it is shown that these release characteristics are exquisitely dependent on the presence of DOPE and cholesterol. In the absence of DOPE, much slower release rates are observed, with maximum release levels of 50% after a 2-h incubation at 20 degrees C. Remarkably, threshold levels of more than 10 mol% cholesterol are required before any appreciable release is observed. [31P]NMR spectroscopy and freeze-fracture electron microscopy studies reveal that systems giving rise to rapid release of doxorubicin exhibit limited formation of inverted hexagonal (H(II)) phase, suggesting that these lipids facilitate drug release by formation of local regions of non-bilayer structure. It is concluded that drug release triggered by mixing anionic and cationic liposomes could be of utility in drug delivery applications.