Arginine-
glycine-
aspartate (RGD) has been shown to possess a strong affinity for the
integrins overexpressed in
tumor cells, especially during
tumor invasion, angiogenesis and metasis. Based on work from others, a novel tetrapeptide,
arginine-
glycine-
aspartate-phenylanaline (RGDF), has been designed and studied as a homing device to direct
liposomal doxorubicin (DOX) to
tumor cells in this work. In order to incorporate RGDF into liposomal DOX preparations, RGDF was conjugated with three different
fatty alcohols to achieve RGDF-
fatty alcohol conjugates.
Glycine-
glycine-
aspartate-phenylanaline (GGDF)-
lauryl alcohol conjugate was synthesized as a negative control. RGDF-
fatty alcohol conjugates (RGDFO(CH(2))(n)CH(3)) and GGDF-
lauryl alcohol conjugate (L-GGDFC12-DOX) incorporated liposomal preparations were obtained by first preparing
liposomes using the film dispersion method followed by loading DOX using a transmembrane pH gradient method. Because of their amphipathic nature, RGDF- or GGDF-
fatty alcohol conjugates are expected to be readily incorporated into
liposomes with their fatty alkanyl chains being intercalated between fatty acyl chains of liposomal bilayers and the hydrophilic
peptide moiety (RGDF or GGDF) being anchored on the surface of
liposomes. The particle size and zeta potential of liposomal DOX preparations containing RGDF-
fatty alcohol conjugate (L-RGDF-DOXs) or L-GGDFC12-DOX were measured, and their morphology was studied using transmission electron microscopy. In vitro DOX release profile from RGDF incorporated liposomal DOX was measured. The antitumor activities of RGDF incorporated liposomal DOX preparations were evaluated in ICR mice inoculated with
sarcoma S(180), which is known to express α(v)β(3)
integrin. Both conventional liposomal DOX preparation (L-DOX) without RGDFO(CH(2))(n)CH(3) and L-GGDFC12-DOX were used as negative controls. Our results showed improved
tumor growth inhibition with L-RGDF-DOXs over
doxorubicin hydrochloride solution, L-DOX and L-GGDFC12-DOX. Pathological examination of
tumor biopsy demonstrated that L-RGDF-DOXs induced enhanced
tumor cell death in comparison to negative controls. Pharmacokinetic studies showed that the concentrations of DOX found in
tumor sites were increased by 1.7-4.5-fold when liposomal DOX preparation containing RGDF-
lauryl alcohol conjugate (L-RGDFC12-DOX) was administered in comparison to when L-GGDFC12-DOX or
doxorubicin hydrochloride solution was administered. The concentrations of DOX found in the heart, which is the main site of toxic effects of DOX, were significantly reduced when L-RGDFC12-DOX was administered in comparison to when L-GGDFC12-DOX or
doxorubicin hydrochloride solution was administered.