Conjugation of Arginine-Glycine-Aspartic Acid Peptides to Poly(ethylene oxide)-b-poly(E-caprolactone) Micelles for Enhanced Intracellular Drug Delivery to Metastatic Tumor Cells Xiao-Bing Xiong, ² Abdullah Mahmud, ² Hasan Uludag ˇ , ²,‡ and Afsaneh Lavasanifar* ,² Faculty of Pharmacy and Pharmaceutical Sciences and Department of Chemical & Materials Engineering, Faculty of Engineering, University of Alberta, Edmonton, Alberta T6G 2N8, Canada Received October 6, 2006; Revised Manuscript Received December 6, 2006 An arginine-glycine-aspartic acid (RGD) containing model peptide was conjugated to the surface of poly(ethylene oxide)-block-poly(ǫ-caprolactone) (PEO-b-PCL) micelles as a ligand that can recognize adhesion molecules overexpressed on the surface of metastatic cancer cells, that is, integrins, and that can enhance the micellar delivery of encapsulated hydrophobic drug into a tumor cell. Toward this goal, PEO-b-PCL copolymers bearing acetal groups on the PEO end were synthesized, characterized, and assembled to polymeric micelles. The acetal group on the surface of the PEO-b-PCL micelles was converted to reactive aldehyde under acidic condition at room temperature. An RGD-containing linear peptide, GRGDS, was conjugated on the surface of the aldehyde-decorated PEO-b-PCL micelles by incubation at room temperature. A hydrophobic fluorescent probe, that is, DiI, was physically loaded in prepared polymeric micelles to imitate hydrophobic drugs loaded in micellar carrier. The cellular uptake of DiI loaded GRGDS-modified micelles by melanoma B16-F10 cells was investigated at 4 and 37 °C by fluorescent spectroscopy and confocal microscopy techniques and was compared to the uptake of DiI loaded valine-PEO-b-PCL micelles (as the irrelevant ligand decorated micelles) and free DiI. GRGDS conjugation to polymeric micelles significantly facilitated the cellular uptake of encapsulated hydrophobic DiI most probably by intergrin-mediated cell attachment and endocytosis. The results indicate that acetal-terminated PEO-b-PCL micelles are amenable for introducing targeting moieties on the surface of polymeric micelles and that RGD- peptide conjugated PEO-b-PCL micelles are promising ligand-targeted carriers for enhanced drug delivery to metastatic tumor cells. Introduction New blood vessels formed in a tumor to provide nutrients and oxygen for growing tumor cells usually have large gaps in their endothelium. This allows the extravasation of nanoparticles to the extravascular space surrounding the tumor cells. The permeated nanocarrier usually gets trapped in the tumor because the lymphatic system that drains fluids out of other organs is absent in tumors. This phenomenon, known as the enhanced permeation and retention (EPR) effect, is believed to be the reason for the passive accumulation of carriers of <200 nm with prolonged blood circulation properties (e.g., polymeric micelles and stealth liposomes) in solid tumors. Polymeric micelles are core/shell structures formed through self-assembly of amphiphilic block copolymers. The nanoscopic dimension as well as unique properties offered by separated core and shell domains of polymeric micelles has made them one of the most promising carriers for drug targeting by EPR in cancer. 1-3 The nanoscopic size of polymeric micelles makes the carrier unrecognizable by the phagocytic cells of the reticuloendothelial system (RES) and prolongs their blood circulation. 4 The small size of polymeric micelles is also expected to facilitate carrier’s extravasation from tumor vas- culature and to ease the penetration of the extravasated carrier within a solid tumor tissue. Finally, a capacity for the stabilized encapsulation of hydrophobic compounds offered by polymeric micelles is considered as one of their advantages over other colloidal systems for targeted delivery of anticancer agents, most of which are hydrophobic. Research in the field of polymeric micellar drug delivery has been expanded tremendously during the past decade. However, achieving high targeting efficiency for hydrophobic drugs by polymeric micelles at the tumor site still remains a major challenge. Despite high accumulation of nanosized micelles in tumor tissue as a result of EPR effect, accumulation of the encapsulated drug at the cellular and molecular drug targets cannot be guaranteed. 5-7 Efficient drug targeting by polymeric micelles in most cases is hampered by either premature drug release from the micellar nanocontainers before the carrier reaches the tumor targets or insufficient intracellular delivery of the encapsulated anticancer drug to the tumor cells. 6 Finding the right polymeric micellar system that can provide a proper balance between the two properties, that is, avoiding premature drug release outside tumor site but promoting cellular internal- ization or obtaining triggered drug release at the tumor site, poses a challenge for efficient targeted drug delivery by polymeric micelles. Several studies suggest that attachment of cell-specific ligands on the surface of colloidal carriers can be used as an efficient strategy to enhance cellular internalization of nanocarriers at desired tissue. 8-11 In this context, covalent attachment of cell- specific ligands, for example, sugars, peptides, and monoclonal antibodies, on the surface of polymeric micellar delivery systems has been pursued to enhance drug delivery to various cells. 12-15 For tumor targeting, cancer-specific peptides are more appropri- ate than monoclonal antibodies because they are smaller and * To whom correspondence should be addressed. Phone: 780-492-2742. Fax: 780-492-1217. E-mail: alavasanifar@pharmacy.ualberta.ca. ² Faculty of Pharmacy and Pharmaceutical Sciences. ‡ Department of Chemical & Materials Engineering. 874 Biomacromolecules 2007, 8, 874-884 10.1021/bm060967g CCC: $37.00 © 2007 American Chemical Society Published on Web 02/22/2007