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.     

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.     

Techniques for encapsulating bioactive agents into liposomes

As a prerequisite for the use of liposomes for delivery of biologically active agents, techniques are required for the efficient and rapid entrapment of such agents in liposomes. Here we review the variety of procedures available for trapping hydrophilic and hydrophobic compounds. Considerations which are addressed include factors influencing the choice of a particular liposomal system and techniques for the passive entrapment of drugs in multilamellar vesicles and unilamellar vesicles. Attention is also paid to active trapping procedures relying on the presence of (negatively) charged lipid or transmembrane ion gradients. Such gradients are particularly useful for concentrating lipophilic cationic drugs inside liposomes, allowing trapping efficiencies approaching 100%.     

Strategies for Optimizing Liposomal Doxorubicin

Abstract

Liposome encapsulation of doxorubicin can dramatically alter its biological activity, resulting in decreased toxicity and equivalent or increased antitumor potency. Since the physical characteristics of the liposome carrier system (size, lipid composition, and lipid dose) can have profound effects on the pharmacologic properties of vesicles administered intravenously, it may be expected that the therapeutic activity of liposomal doxorubicin will be sensitive to these properties. To determine the influence of these variables on the toxicity and efficacy properties of liposomal doxorubicin, transmembrane pH gradient-dependent active encapsulation techniques have been utilized to generate liposomal doxorubicin preparations in which the vesicle size, lipid composition, and drug to lipid ratio can be independently varied. these studies indicate that the toxicity of liposomal doxorubicin is related to the stability of the preparation in the circulation. This property is dictated primarily by vesicle lipid composition, although the drug to lipid ratio can also exert an influence. In contrast, the antitumor activity of liposomal doxorubicin appears most sensitive to the size of the vesicle system. Specifically, antitumor drug potency increases as the vesicle size is decreased. these studies demonstrate that manipulating the physical characteristics of liposomal anticancer pharmaceuticals can lead to preparations with optimized therapeutic activity.     

Formation of transition metal-doxorubicin complexes inside liposomes

Doxorubicin complexation with the transition metal manganese (Mn(2+)) 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 Mn(2+) 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, MnSO(4) or MnCl(2). In response to a pH gradient or a Mn(2+) 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 Mn(2+) 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 Mn(2+) 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 Mn(2+) 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.     

Uptake of adriamycin into large unilamellar vesicles in response to a pH gradient

Previous work has shown that adriamycin can be accumulated into large unilamellar vesicle (LUV) systems in response to K⁺ diffusion potential established by valinomycin. It is demonstrated here that adriamycin can also be rapidly and efficiently accumulated into egg phosphatidylcholine (egg PC) and egg PC-cholesterol (1:1) LUVs in response to a transmembrane pH gradient (interior acidic) in the absence of ionophores. This ‘active’ loading gives rise to trapping efficiencies as high as 98%, interior drug concentrations as high as 100 mM and significantly enhances drug retention within the vesicles. This procedure may be of general utility for loading liposomal systems for in vivo drug delivery.     

Influence of ion gradients on the transbilayer distribution of dibucaine in large unilamellar vesicles

The uptake of dibucaine into large unilamellar vesicles in response to proton gradients (delta pH; inside acidic) or membrane potentials (delta psi; inside negative) has been investigated. Dibucaine uptake in response to delta pH proceeds rapidly in a manner consistent with permeation of the neutral (deprotonated) form of the drug, reaching a Henderson-Hasselbach equilibrium where [dibucaine]in/[dibucaine]out = [H+]in/[H+]out and where the absolute amount of drug accumulated is sensitive to the buffering capacity of the interior environment. Under appropriate conditions, high absolute interior concentrations of the drug can be achieved (approximately 120 mM) in combination with high trapping efficiencies (in excess of 90%). Dibucaine uptake in response to delta psi proceeds more than an order of magnitude more slowly and cannot be directly attributed to uptake in response to the delta pH induced by delta psi. This induced delta pH is too small (less than or equal to 1.5 pH units) to account for the transmembrane dibucaine concentration gradients achieved and does not come to electrochemical equilibrium with delta psi. Results supporting the possibility that the charged (protonated) form of dibucaine can be accumulated in response to delta psi were obtained by employing a permanently positively charged dibucaine analogue (N-methyldibucaine). Further, the results suggest that delta psi-dependent uptake may depend on formation of a precipitate of the drug in the vesicle interior. The uptake of dibucaine into vesicles in response to ion gradients is of direct utility in drug delivery and controlled release applications and is related to processes of drug sequestration by cells and organelles in vivo.     

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.     

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.     

Encapsulation of Vincristine in Liposomes Reduces its Toxicity and Improves its Anti-Tumor Efficacy

Abstract

Vincristine is a potent therapeutic agent with activity against a variety of tumor types. It is a cell-cycle specific agent which has exhibited enhanced anti-tumor activity when delivered in liposomal form. Vincristine can be encapsulated into large unilamellar vesicles in response to a transmembrane pH gradient with trapping efficiencies approaching 100%. The extent of vincristine encapsulation, and the subsequent retention of the drug within the liposomes, both in vitro and in vivo, are strongly dependent on the lipid composition of the liposome and on the magnitude of the transmembrane pH gradient. Liposomal formulations of vincristine have been optimized for both liposome circulation longevity, drug retention characteristics and in vivo antitumor activity. When compared to free vincristine, these formulations significantly increase the levels of vincristine remaining in the plasma after i.v. administration. These formulations also significantly increase the delivery of vincristine to tumor sites. As a consequence of the improved accumulation of vincristine at tumor sites, liposomal formulations of vincristine exhibit dramatically improved efficacy against a variety of ascitic and solid murine and human tumors than does free vincristine. Liposomal vincristine is expected to be of wide utility in a variety of human malignancies.     

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.     

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.     

Influence of drug-to-lipid ratio on drug release properties and liposome integrity in liposomal doxorubicin formulations

Recent studies have shown that the release properties of vincristine encapsulated in large unilamellar vesicles (LUV) can be regulated by varying the drug-to-lipid (D/L) ratio. In this work it is shown that the drug-to-lipid ratio technique for regulating drug release also applies to doxorubicin encapsulated in LUV. In particular it is shown that the half-times (T(1/2)) for doxorubicin release from distearoylphosphatidylcholine (DSPC)/cholesterol LUV in vitro can be increased more than six-fold by increasing the D/L ratio from 0.05 (wt/wt) to 0.39 (wt/wt). This behavior is consistent with the behavior expected for drugs that precipitate following accumulation into liposomes. It is shown that the release properties of ciprofloxacin--a drug that does not precipitate following accumulation into LUV--are not affected by the D/L ratio. It is also shown that the crystalline intravesicular doxorubicin precipitates observed as the D/L ratio is raised from 0.05 to 0.46 adopt increasingly unusual morphologies. Linear crystals are observed at lower D/L values, however triangular and rectangular variations are observed as the D/L ratio is increased, and induce considerable distortion in vesicle morphology. It is noted that trapping efficiency following uptake of external doxorubicin into LUV is reduced from nearly 100% at a D/L ratio of 0.05 (wt/wt) to less than 70% at an (initial) D/L ratio of 0.8 (wt/wt). It is suggested that this arises, at least in part, from membrane-disrupting effects of internal drug crystals as they increase in size.     

Influence of Drug-to-Lipid Ratio on Drug Release Properties and Liposome Integrity in Liposomal Doxorubicin Formulations

Recent studies have shown that the release properties of vincristine encapsulated in large unilamellar vesicles (LUV) can be regulated by varying the drug-to-lipid (D/L) ratio. In this work it is shown that the drug-to-lipid ratio technique for regulating drug release also applies to doxorubicin encapsulated in LUV. In particular it is shown that the half-times (T_1/2) for doxorubicin release from distearoylphosphatidylcholine (DSPC)/cholesterol LUV in vitro can be increased more than six-fold by increasing the D/L ratio from 0.05 (wt/wt) to 0.39 (wt/wt). This behavior is consistent with the behavior expected for drugs that precipitate following accumulation into liposomes. It is shown that the release properties of ciprofloxacin—a drug that does not precipitate following accumulation into LUV—are not affected by the D/L ratio. It is also shown that the crystalline intravesicular doxorubicin precipitates observed as the D/L ratio is raised from 0.05 to 0.46 adopt increasingly unusual morphologies. Linear crystals are observed at lower D/L values, however triangular and rectangular variations are observed as the D/L ratio is increased, and induce considerable distortion in vesicle morphology. It is noted that trapping efficiency following uptake of external doxorubicin into LUV is reduced from nearly 100% at a D/L ratio of 0.05 (wt/wt) to less than 70% at an (initial) D/L ratio of 0.8 (wt/wt). It is suggested that this arises, at least in part, from membrane-disrupting effects of internal drug crystals as they increase in size.     

Uptake of antineoplastic agents into large unilamellar vesicles in response to a membrane potential

Many drugs exhibit lipophilic and cationic (basic) characteristics. Previous studies have shown that lipophilic cations can be accumulated into model membrane 'liposomal' (vesicular) systems in response to establishing a membrane potential (inside negative) across the vesicle membrane. We demonstrate here that the anticancer drugs, adriamycin and vinblastine, can be rapidly accumulated into egg phosphatidylcholine large unilamellar vesicles in response to a valinomycin-dependent K+ diffusion potential (delta psi) to achieve high effective interior concentrations. Further, trapping efficiencies approaching 100% can be easily achieved. The influence of lipid composition and the requirement for valinomycin have been examined for adriamycin. Equimolar cholesterol levels inhibit the uptake process at 20 degrees C. However, incubation at higher temperature results in enhanced uptake. Similarly, the presence of egg phosphatidylserine or incubation at elevated temperatures results in significant adriamycin uptake in the absence of valinomycin. It is shown that the adriamycin retention time in the vesicles is enhanced by an order of magnitude or more when actively trapped by the presence of a membrane potential in comparison to passive trapping procedures. It is suggested that such active trapping procedures may be of use for loading liposomal systems for drug delivery applications, and may provide avenues for controlled release of encapsulated material.     

Development of stealth liposome coencapsulating doxorubicin and fluoxetine

Stealth liposomes form an important subset of liposomes, demonstrating prolonged circulation half-life and improved safety in vivo. Caelyx? (liposomal doxorubicin; Merck & Co., Whitehouse Station, New Jersey, USA) is a successful example of the application of stealth liposomes in anticancer treatment. However, multidrug resistance (MDR) to chemotherapy still remains a critical problem, accounting for more than 90% of treatment failure in patients with advanced cancer. To circumvent MDR, fluoxetine and doxorubicin were tested in combination for synergistic activity in MCF-7 (human breast carcinoma) and MCF-7/adr (doxorubicin-resistant human breast carcinoma) cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell-viability assay. Coencapsulation of doxorubicin and fluoxetine, using an ammonium sulphate gradient, was investigated, and a factorial experiment was designed to determine the optimal drug-to-lipid (D/L) ratio for coencapsulation. Drug release from Dox-Flu-SL (stealth liposome coencapsulating doxorubicin and fluoxetine) under both in vitro and in vivo conditions was determined. In MCF-7 cells, synergism was demonstrated at specific doxorubicin:fluoxetine ratios of between 0.09 and 0.5 (molar ratio), while MCF/7/adr cells demonstrated synergism across all drug ratios. Coencapsulation of doxorubicin and fluoxetine (Dox-Flu-SL) was successfully achieved (optimal doxorubicin:fluoxetine:lipid molar ratio of 0.02:0.05:1), obtaining a mean concentration of 257 ± 12.1 and 513 ± 29.3 μM for doxorubicin and fluoxetine, respectively. Most important, Dox-Flu-SL demonstrated drug release in synergistic ratios in cell-culture media, accounting for the improved cytotoxicity of Dox-Flu-SL over liposomal doxorubicin (LD) in both MCF-7 and MCF-7/adr cells. Pharmacokinetic studies also revealed that Dox-Flu-SL effectively prolonged drug-circulation time and reduced tissue biodistribution. Dox-Flu-SL presents a promising anticancer formulation, capable of effective reversal of drug resistance, and may constitute a novel approach for cancer therapy.     

Nanocapsules: lipid-coated aggregates of cisplatin with high cytotoxicity.

Cisplatin is one of the most widely used agents in the treatment of solid tumors, but its clinical utility is limited by toxicity. The development of less toxic, liposomal formulations of cisplatin has been hampered by the low water solubility and low lipophilicity of cisplatin, resulting in very low encapsulation efficiencies. We describe a novel method allowing the efficient encapsulation of cisplatin in a lipid formulation; it is based on repeated freezing and thawing of a concentrated solution of cisplatin in the presence of negatively charged phospholipids. The method is unique in that it generates nanocapsules, which are small aggregates of cisplatin covered by a single lipid bilayer. The nanocapsules have an unprecedented drug-to-lipid ratio and an in vitro cytotoxicity up to 1000-fold higher than the free drug. Analysis of the mechanism of nanocapsule formation suggests that the method may be generalized to other drugs showing low water solubility and lipophilicity.     

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.     

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.