The experimental design as practical approach to develop and optimize a formulation of peptide-loaded liposomes

To investigate the encapsulation of Print 3G, a peptidic agent that could reduce
the angiogenic development of breast tumors, pegylated liposomes used as
intravenous vectors were studied and characterized. Recently, the path of
liposomes has been explored with success to improve the pharmacological
properties of peptidic drugs and to stabilize them. In this study, loaded
unilamellar vesicles composed of SPC:CHOL:mPEG2000-DSPE (47:47:6)
were prepared by the hydration of lipid film technique. An HPLC method was
developed and validated for the determination of Print 3G to calculate its
encapsulation efficiency. Observed Print 3G adsorption on different materials
employed during liposome preparation (such as glass beads, tubing, and
connections for extrusion) led to the modification of the manufacturing method.
The freeze-thawing technique was used to enhance the amount of Print 3G
encapsulated into blank liposomes prepared using the hydration of lipid film
procedure. Many factors may influence peptide entrapment, namely the number
of freeze-thawing cycles, the lipid concentration, the peptide concentration, and
the mixing time. Consequently, a design of experiments was performed to
obtain the best encapsulation efficiency while minimizing the number of
experiments. The lipid concentration and the number of freeze-thawing cycles
were identified as the positive factors influencing the encapsulation. As a result
of the optimization, an optimum was found and encapsulation efficiencies were
improved from around 30% to 63%. Liposome integrity was evaluated by
photon correlation spectroscopy and freeze-fracture electron microscopy to
ensure that the selected formulation possesses the required properties to be a
potential candidate for further in vitro and in vivo experiments.