Method of drug loading in liposomes by gradient

A method for preparing a pharmaceutical composition comprising:

• (a) contacting a solution of liposomes with a pharmaceutical agent in an aqueous solution of at least about 60 mM of an acid, at a temperature wherein the protonated form of the pharmaceutical agent is charged and is not capable of permeating the membrane of the liposomes, and wherein the unprotonated form of the pharmaceutical agent is uncharged and is capable of permeating the membrane of the liposomes;
• (b) cooling the solution to a temperature at which the unprotonated form of the pharmaceutical agent is not capable of permeating the membrane of the liposomes;
• (c) contacting the solution with a weak base, in an amount effective to raise the pH of the internal liposome to provide gradient loaded liposomes having a lower inside/higher outside pH gradient;
• and
• (d) combining the liposomes with a pharmaceutically acceptable carrier to provide the pharmaceutical composition.

A gradient loaded liposome having a lower inside/higher outside pH gradient prepared by the process comprising:

• (a) contacting a solution of liposomes with a pharmaceutical agent in an aqueous solution of at least about 60 mM of an acid, at a temperature wherein the protonated form of the pharmaceutical agent is charged and is not capable of permeating the membrane of the liposomes, and wherein the unprotonated form of the pharmaceutical agent is uncharged and is capable of permeating the membrane of the liposomes;
• (b) cooling the solution to a temperature at which the unprotonated form of the pharmaceutical agent is not capable of permeating the membrane of the liposomes;
• and
• (c) contacting the solution with a weak base, in an amount effective to raise the pH of the internal liposome to provide gradient loaded liposomes having a lower inside/higher outside pH gradient.