Recently, a large number of investigations have been conducted on drug delivery system (DDS) for delivering and distributing a drug to the target lesion site at high efficiency with high safety. One such approach has been use of a closed vesicle such as liposome, emulsion, lipid microsphere, or nano particle for the transporter (carrier) of the drug. Use of such closed vesicle in making the DDS into practical application, however, is associated with various problems which need to be overcome, and critical challenges have been escape from the biological recognition mechanism for a foreign substance and in vivo control of the pharmacokinetics. In other words, delivery of the closed vesicle to the target site at high selectivity is achieved only when the stability of the closed vesicle in the blood is secured by eliminating capturing of the closed vesicle in the reticuloendothelial system (RES) such as liver and spleen and avoiding aggregation of the closed vesicle by the interaction (adsorption) with opsonin protein or plasma protein in the blood.
One known method of obviating such problems is modification of the membrane with a hydrophilic macromolecule. Closed vesicle, and in particular, liposomes modified with a hydrophilic macromolecule have been developed for use in clinical practice since they have realized high retentivity in blood, and hence, passive accumulation in the tissues having an increased vascular permeability, for example, in the tumor tissue or in the inflammatory site (see Patent Documents 1 to 3 and Non-patent Documents 3 to 5). A typical preferable modifier used in such modification with the hydrophilic practice is a derivative of polyethylene glycol (PEG) produced by attaching a lipid such as a phospholipid or a cholesterol to the polyethylene glycol. An example of the commercially available versatile modifier is a derivative of polyethylene glycol produced by attaching a phospholipid such as diacylphosphatidyl ethanolamine to the polyethylene glycol.
The liposome which is subject to such modification is a closed vesicle formed from a lipid bilayer having an aqueous phase (an internal aqueous phase) encapsulated in its interior. Various liposomes having different layer structures are known in the art, and exemplary liposomes include unilamellar vesicles such as a small unilamellar vesicle (SUV) and a large unilamellar vesicle comprising a single layer of a lipid bilayer and a multilamellar vesicle (MLV) comprising two or more layers. Use of a liposome with a MLV structure for prevention of leakage of the encapsulated drug therein have been proposed (see Patent Document 4).
Encapsulation of the drug in the liposome as described above may be accomplished by various methods, and a method known in the art capable of encapsulating the drug at a high concentration is the method using ion gradient such as pH gradient (see Patent Documents 5 to 7 and Non-patent Document 6).    [Patent Document 1] JP-5-505173-A    [Patent Document 2] JP-7-20857-B    [Patent Document 3] JP-2667051-B    [Patent Document 4] WO 01/000173    [Patent Document 5] U.S. Pat. No. 5,077,056    [Patent Document 6] JP-2847065-B    [Patent Document 7] JP-2659136-B    [Non-patent Document 1] Cancer Lett., 1997, 118(2), p. 153    [Non-patent Document 2] Br. J. Cancer., 1997, 76(1), p. 83    [Non-patent Document 3] D. D. Lasic, “LIPOSOMES from Physics to Applications”, Elsevier, 1993    [Non-patent Document 4] Martin C. Woodle and Gerrit Storm ed., “Long Circulating Liposomes: Old Drugs, New Therapeutics”, Springer, 1997    [Non-patent Document 5] D. D. Lasic and D. Papahadjopoulos ed., “Medical Applications of LIPOSOMES”, Elsevier, 1998    [Non-patent Document 6] G. Gregoriadis ed., “Liposome Technology Liposome Preparation and Related Techniques” 2nd edition, Vol. I-III, CRC Press