Method for preparing transmembrane pH-gradient vesicles

A method for preparing transmembrane pH-gradient vesicles, comprising the steps of:

• a) preparing vesicles made from at least one matrix substance in an aqueous medium having an osmolarity of not more than 200 mOsm/l and a pH in the range of 6.0 to 7.5, wherein the matrix substance is chosen from the group consisting of amphiphilic lipids and amphiphilic block copolymers, sterilizing the vesicles, and, optionally, storing the sterilized vesicles,
• b) mixing the sterilized vesicles with a sterile basic or acidic buffer having an osmolarity being at least 200 mOsm/l higher than the osmolarity of the aqueous medium of step a) to apply an osmotic shock to the vesicles and to obtain buffer-filled vesicles, wherein the basic or acidic buffer is added in an amount sufficient to cause temporary destabilization of the vesicles in step b), and
• c) diluting a mixture of the aqueous medium and the basic or acidic buffer containing the buffer-filled vesicles by adding a sterile neutralizing solution to obtain sterile transmembrane pH-gradient vesicles suspended in a sterile suspension buffer, wherein the suspension buffer differs from the basic or acidic buffer in pH value, and wherein the neutralizing solution does not destabilize the vesicles in step c).