Anti-tumor efficacy of tumor vasculature-targeted liposomal doxorubicin
Introduction
Angiogenesis, a term describing the process of new blood vessel formation, is an essential process in the growth and metastasis of a tumor. It is a response to a shift in balance between pro- and anti-angiogenic mediators in malignant tissue. The resulting pro-angiogenic state favors the formation of new blood vessels [1], [2]. Given the dependency of tumor growth on angiogenesis, disrupting this process may be a promising treatment strategy [3], [4]. A drug delivery system specifically targeting cells involved in the angiogenic process could be a valuable tool to reach this goal.
Endothelial cells are crucial in angiogenesis [5], [6]. Many anti-angiogenic strategies are aimed at inhibiting the proliferation or motility of the endothelium [3]. Alpha v beta 3-integrins are overexpressed on actively proliferating endothelium in and around tumor tissue [7], [8]. These integrins may therefore represent a promising target for drug delivery. Small peptide ligands for the alpha v-integrins have been identified that share an RGD-motif as a consensus sequence. RGD-peptides that are constrained in a preferred cyclic conformation show an increased affinity for integrin interaction [9]. Multivalency, e.g. by display of the ligand on the surface of a drug delivery system, may further increase the affinity of the interaction with the target cells [10].
By coupling cyclic RGD-peptides to the distal end of poly(ethylene glycol)-coated long-circulating liposomes (LCL), we aimed at obtaining a stable, long-circulating drug delivery system functioning as a platform for multivalent interaction with alpha v beta 3-integrins. In this study, in vitro binding to human umbilical vein endothelial cells was evaluated as well as in vivo binding in a mouse tumor model. Therapeutic efficacy was investigated in a doxorubicin-insensitive murine C26 colon carcinoma model. An insensitive tumor model was chosen because anti-tumor effects observed in such a model are unlikely to arise from direct cytotoxic effects on tumor cells, but rather from inhibition of other cell types necessary for tumor growth.
Section snippets
Preparation of liposomes
Dipalmitoylphosphatidylcholine (Lipoid, Ludwigshafen, Germany), cholesterol (Sigma, St. Louis, MO, USA), PEG-2000 distearoylphosphatidylethanolamine (Lipoid) and maleimide-PEG-2000 distearoylphosphatidylethanolamine (Shearwater Polymers, Huntsville, AL, USA), were dissolved in chloroform:methanol (2:1, v/v) in a round-bottom flask in a molar ratio of 1.85:1.0:0.075:0.075, respectively. 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD) (0.1 mol.% ) (Molecular Probes,
FACS analysis of liposome interaction with HUVEC
HUVEC were incubated with DiD-labeled LCL, RAD–LCL, or RGD–LCL. RGD–LCL exhibited a sixfold higher level of association to HUVEC compared to RAD–LCL and LCL (P<0.01) (Fig. 2). The latter two liposome preparations hardly interacted with HUVEC. The higher degree of association of the RGD-modified LCL is the result of the coupling of the RGD-peptide to the distal end of the PEG-chains in the LCL. Multivalent display of RGD-targeting ligands has been shown earlier to result in a high endothelial
Conclusions
Coupling of RGD to LCL targets these liposomes to angiogenic endothelial cells in vitro and in vivo. Doxorubicin-containing RGD–LCL were able to decelerate tumor growth in a doxorubicin-insensitive tumor model. In this model, LCL-encapsulated doxorubicin failed to inhibit tumor growth. Likely, the superior therapeutic efficacy of RGD–LCL is the result of inhibition of tumor progression via inhibition of angiogenesis rather than via direct cytotoxic effects on tumor cells.
Acknowledgements
This study was funded by grant UU 2000-2185 of the Dutch Cancer Society.
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