Her2-targeted pegylated liposomal doxorubicin: retention of target-specific binding and cytotoxicity after in vivo passage.
BACKGROUND
Receptor-directed targeting of ligand-bearing liposomes to tumor cells may enhance therapeutic efficacy by intracellular delivery of a concentrated payload of liposomal drug. The goal of this study was to assess whether Her2-targeted pegylated liposomal doxorubicin (PLD) retains its binding ability to Her2-expressing target cells through circulation in the blood and extravasation to tumor interstitial fluid.


METHODS
PLD was grafted with a lipophilic conjugate of an anti-Her2 scFv antibody fragment at an approximate ratio of 7.5, 15, or 30 ligands per liposome. BALB/c mice were injected with J6456 lymphoma cells into the peritoneal cavity to generate malignant ascites used as a model for tumor interstitial fluid. When abdominal swelling developed, Her2-targeted (HT-) PLD and non-targeted PLD were injected into the mice i.v. at a dose of 15 mg/kg. The ascitic fluid was collected 48 h later, ascitic tumor cells were removed, and the doxorubicin levels in the cell-free ascitic fluid and plasma were determined. Binding of the cell-free ascitic fluid was tested in vitro against two Her2-expressing human tumor cell lines (N87, SKBR-3) and compared to the binding of shelf formulations (not passaged in vivo) of HT-PLD and PLD, by measuring the amount of cell-associated doxorubicin.


RESULTS
Plasma and ascitic fluid levels of HT-PLD were only slightly below those of PLD indicating that, the Her2 ligand did not cause any significant change in the clearance rate of PLD. The in vitro binding of HT-PLD containing ascitic fluid to Her2-expressing cells was increased 10 to 20-fold above that of PLD-containing ascitic fluid, similarly to the 20-fold difference in binding between shelf Her2-PLD and PLD. The in vitro cytotoxicity of ascitic fluid containing HT-PLD tested against Her2-expressing tumor cells was far greater than that of PLD, and similar to that of the shelf formulations, indicating that the selective pharmacological activity of HT-PLD is preserved after in vivo passage. Optimal results were obtained with HT-PLD formulated with 15 ligands per liposome.


CONCLUSIONS
HT-PLD retains most of its original binding capacity to Her2-expressing cells after in vivo passage indicating that the ligand is stably maintained in vivo in association with the doxorubicin liposomal carrier, and confirming the validity of the post-formulation ligand grafting approach for liposome targeting. Targeting of PLD using a Her2 antibody fragment provides an important means of in vivo selective drug delivery to tumors expressing the Her2 receptor.
Shmeeda
Hilary
H
Experimental Oncology Laboratory, Shaare Zedek Medical Center, Jerusalem, Israel.
Tzemach
Dina
D
Mak
Lidia
L
Gabizon
Alberto
A