Doxil — The First FDA-Approved Nano-Drug: From an Idea to a Product - Harnessing Biomaterials in Nanomedicine: Preparation, Toxicity, and Applications - page 345

liposomal membranes. Diphosphatidyl glycerol (cardiolipin, DPG),
phosphatidyl glycerol (PG), and phosphatidylserine (PS) were all
active in improving the encapsulation, with DPG encapsulation
being the best in terms of drug association, in agreement with
the highest association constant of DPG doxorubicin of 1.6 × 10 -6 ,
compared with 1.8 × 10 -4 for PS and ~0.0 for PC (Goormarghitigh
and Ruysschaert, 1984). Exposure to serum (without large dilution!)
did not cause significant doxorubicin release. For practical reasons
such as DPG availability and cost (at that time), as well as potential
immunogenicity, we excluded the DPG from our studies.
Another important aspect of this research was that unilamellar
vesicles (either SUV < 100 nm or LUV > 100 nm in diameter) capture
much less drug and lose their drug content faster than vesicles of the
same lipid composition in larger sizes and lamellarity (Goren et al.,
1990; Goren, 1990) including either large (>100 nm) oligolamellar
(OLV) or multilamellar large vesicles (MLV). Large and small are
functional definitions that describe vesicles in which the external two
leaflets of the external lipid bilayer have diff erent (SUV) or almost
the same (OLV and MLV) curvature (Lichtenberg and Barenholz,
1988). This was another warning signal that could predict a failure
in humans, which we did not understand in the early 1980s.
Finally, we found that formulations composed of saturated lipids
have significantly lower drug capture than the formulations that
are based on phospholipids of the identical headgroups but having
unsaturated hydrocarbon chains. Namely, unsaturated phospholipids
seemed to be superior to saturated phospholipids. This may be
explained by the larger free volume in the membrane composed of
unsaturated lipids, which leaves more space for the drug.
With both PS- and PG-containing OLV (as the negatively charged
lipid), we achieved a similar drug to lipid ratio over the broad
range of 0.05-0.26 (depending on fine tuning, Amselem et al., 1990,
1992), but as PS is less chemically stable and more expensive, PG
became our negatively charged lipid of choice. The formulation was
optimized for various variables, of which level of PG (demonstrated
in Figure 12.2) was a main one.
The result of all these optimization processes was our preferred
formulation for OLV-DOX, a liposome 300-500 nm in diameter. It
was composed of egg PC : egg PG : cholesterol (7 : 3 : 4 mole ratio)
and was fabricated by extrusion (Amselem et al., 1989-90, 1990,
1992). Due to the use of egg-derived unsaturated phospholipids