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.

The accumulation of drugs within large unilamellar vesicles exhibiting a proton gradient: a survey.

We have shown previously that transmembrane proton gradients can be used to efficiently accumulate biogenic amines [M.B. Bally et al. (1988) Chem. Phys. Lipids 47, 97-107] and doxorubicin [L.D. Mayer, M.B. Bally and P.R. Cullis (1986) Biochim. Biophys. Acta 857, 123-126] to high concentrations within liposomes. To determine the generality of this loading procedure, representative drugs from a variety of different classes (antineoplastics, local anaesthetics, antihistamines, etc.) were examined as to their ability to redistribute in response to a proton gradient. While the majority of drugs examined, all of which are weak bases, were accumulated by large unilamellar vesicles exhibiting a pH gradient (interior acid) the extent of uptake varied considerably between different pharmaceuticals. These differences are discussed in the context of various factors which will likely influence drug accumulation including its membrane/water partition coefficient and its solubility in the intravesicular medium.

Influence of vesicle size, lipid composition, and drug-to-lipid ratio on the biological activity of liposomal doxorubicin in mice.

The effects of vesicle size, lipid composition, and drug-to-lipid ratio on the biological activity of liposomal doxorubicin in mice have been investigated using a versatile procedure for encapsulating doxorubicin inside liposomes. In this procedure, vesicles exhibiting transmembrane pH gradients (acidic inside) were employed to achieve drug trapping efficiencies in excess of 98%. Drug-to-lipid ratios as high as 0.3:1 (wt:wt) could be obtained in a manner that is relatively independent of lipid composition and vesicle size. Egg phosphatidylcholine (EPC)/cholesterol (55:45; mol/mol) vesicles sized through filters with a 200-nm pore size and loaded employing transmembrane pH gradients to achieve a doxorubicin-to-lipid ratio of 0.3:1 (wt/wt) increased the LD50 of free drug by approximately twofold. Removing cholesterol or decreasing the drug-to-lipid ratio in EPC/cholesterol preparations led to significant decreases in the LD50 of liposomal doxorubicin whereas, the LD50 increased 4- to 6-fold when distearoylphosphatidylcholine was substituted for EPC. The results suggest that the stability of liposomally entrapped doxorubicin in the circulation is an important factor in the toxicity of this drug in liposomal form. In contrast, the antitumor activity of liposomal doxorubicin is not influenced dramatically by alterations in lipid composition. Liposomal doxorubicin preparations of EPC, EPC/cholesterol (55:45; mol:mol), EPC/egg phosphatidylglycerol (EPG)/cholesterol (27.5:27.5:45; mol:mol), and distearoylphosphatidylcholine/cholesterol (55:45; mol:mol) all demonstrated similar efficacy to that of free drug when given at doses of 20 mg/kg and below. Higher dose levels of the less toxic formulations could be administered, leading to enhanced increases in life span (ILS) values. Variations in vesicle size, however, strongly influenced the antitumor activity of liposomal doxorubicin. At a dose of 20 mg/kg, large EPC/cholesterol systems are significantly less effective than free drug (with ILS values of 65% and 145%, respectively). In contrast, small systems sized through filters with a 100-nm pore size are more effective than free drug, resulting in an ILS of 375% and a 30% long term (greater than 60 days) survival rate when administered at a dose of 20 mg/kg. Similar size-dependent effects are observed for distearoylphosphatidylcholine/cholesterol systems.