Release of hydrophobic molecules from polymer micelles into cell membranes revealed by Förster resonance energy transfer imaging

Abstract

It is generally assumed that polymeric micelles, upon administration into the blood stream, carry drug molecules until they are taken up into cells followed by intracellular release. The current work revisits this conventional wisdom. The study using duallabeled micelles containing fluorescently labeled copolymers and hydrophobic fluorescent probes entrapped in the polymeric micelle core showed that cellular uptake of hydrophobic probes was much faster than that of labeled copolymers. This result implies that the hydrophobic probes in the core are released from micelles in the extracellular space. Fö rster resonance energy transfer (FRET) imaging and spectroscopy were used to monitor this process in real time. A FRET pair, DiIC 18(3) and DiOC18(3), was loaded into monomethoxy poly(ethylene glycol)-block-poly(D,L-lactic acid) micelles. By monitoring the FRET efficiency, release of the core-loaded probes to model membranes was demonstrated. During administration of polymeric micelles to tumor cells, a decrease of FRET was observed both on the cell membrane and inside of cells, indicating the release of core-loaded probes to the cell membrane before internalization. The decrease of FRET on the plasma membrane was also observed during administration of paclitaxel-loaded micelles. Taken together, our results suggest a membrane-mediated pathway for cellular uptake of hydrophobic molecules preloaded in polymeric micelles. The plasma membrane provides a temporal residence for micelle-released hydrophobic molecules before their delivery to target intracellular destinations. A putative role of the PEG shell in the molecular transport from micelle to membrane is discussed. Block copolymer micelles (1-4) have attracted growing interest as carriers to deliver drugs including antitumor reagents to the target site via the enhanced permeability and retention effect (5). Hydrophobic molecules, such as paclitaxel (PTX), can be incorporated into the core of polymeric micelles by physical entrapment. The core composed of hydrophobic chains is expected to regulate the retention and the release of loaded drug molecules on an appropriate time scale (6-8). With micelle-based drug formulations reaching clinical trials (9, 10), the impetus for understanding the pathways involved in micellar drug delivery starts to emerge. It is thought that intact polymeric micelles are taken into cells, followed by intracellular release of drug molecules (8). Kabanov's group (11) reported that pluronic copolymer micelles could be internalized by an endocytic pathway and increase drug absorption by inhibition of P-glycoprotein drug efflux system in cancer cells. Allen et al. (12) suggested that poly(ethylene oxide)-poly(-caprolactone) (PEO-PCL) micelles enter PC12 cells via endocytosis. To directly monitor the micelles, Luo et al. (13) and Savic et al. (14) chemically conjugated a rhodamine molecule to the end of the PCL block and investigated the internalization of PEO-PCL micelles into P19 and PC12 cells. In their study, micelles were reported to en...

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