Non-PEGylated doxorubicin liposome comes in a three-vial kit and has a lipid composition and liposome size similar to commercial Myocet. Doxorubicin drug cannot be pre-loaded to the liposomes in the same way that is pre-loaded into the PEGylated liposomes. The matrix liposome in PEGylated liposome is hydrogenated soy PC which is a saturated lipid and creates a tight packed liposome suitable for pre-loading and holding the drug inside the liposomes. The matrix lipid in this formulation is L–phosphatidylcholine which is an unsaturated lipid. It makes a loose packed membrane and therefore, it cannot hold the drug for a long time. The doxorubicin drug should be loaded into the liposomes moments before use. The kit comes in three vials. The first vial contains a very concentrated solution of liposomes. The liposomes are made in citrate buffer at pH 4. The second vial is composed of sodium carbonate at pH 11.4. Adding vial 2 to vial 1 makes a pH gradient around the lipid membrane. Due to the difference in the pH inside and outside of the liposomes, there will be a constant flux of proton from the inside to the outside of the liposomes. Due to this flux, doxorubicin molecules move in the opposite direction from the outside to the inside of the liposomes. During the remote loading process, due to pH gradient, the doxorubicin drug is able to freely pass into the liposomes where it is bound by hydrogen and nanocrystals of doxorubicin citrate salt to form larger doxorubicin compounds. As the new doxorubicin complexes are formed, they come together to form one of the three different shapes inside the liposomes. A long singular band that stretches the membrane, a closed circular band or an opened U-shaped band. The circular and U-shaped complexes are the most common ones. Doxorubicin liposomes that are formed by remote loading using pH gradient should be used immediately. PEGylated formulation of doxorubicin liposomes is also available. For more information see here.