Liposomes as nanocarriers of lipid-conjugated antitumor drugs melphalan and methotrexate

Abstract

The inclusion of antitumor agents into nanodimensional carriers such as 100- to 150-nm liposomes makes it possible to reduce the general toxicity of chemotherapeutic drugs by decreasing the free drug concentration in the blood flow and by increasing the passive transport and accumulation of nanocarriers in tumors due to enhanced permeability of defective capillary vessel walls. On the other hand, biodegradable lipid-drug conjugates (lipophilic prodrugs) possess improved pharmacokinetics. In this study, we have determined detailed characteristics of the liposomal formulations of the antitumor drugs melphalan and methotrexate conjugated to rac-1,2-dioleoylglycerol, which contain the drugs in amounts (∼4 mM) sufficient for systemic injections into experimental animals. The liposomes were prepared from a mixture of natural phospholipids and prodrugs (phosphatidylcholine-phosphatidylinositol-prodrug molar ratio, 8 : 1 : 1) by the standard method of extrusion through polycarbonate membranes with 100-nm pores. The liposome size, lamellarity, and degree of aggregation were determined by methods of dynamic (laser) light scattering and transmission electron microscopy (using negative contrasting and freeze fracture techniques), while the liposome composition was checked using gel chromatography with subsequent UV spectrophotometry. It has been established that both diglyceride lipid-drug conjugates are completely included into unilamellar liposomes (bounded by a single lipid bilayer) with an average size of 50–150 nm and this dispersion can be stored for several days without any signs of significant aggregation. The possibility of obtaining liposomal preparations for long-term storage has been studied. It is demonstrated that liposomal drug dispersions can be subjected to deep freezing in liquid nitrogen and then stored for a long period of time at −70°C. For subsequent usage, the dispersion should be defrozen and treated for a short time in an ultrasonic bath, which completely restores the composition and size of the initial particles. Experiments in vitro have shown that a liposomal methotrexate conjugate is capable of overcoming tumor cell resistance to the drug, which is related to impaired transmembrane transport. The drug resistance of human leukemia cells related to a decreased activity of a transport protein (reduced folate carrier) has been decreased 114 times for methotrexate conjugate in liposomal formulation compared to the initial drug.