Cellular Internalization

Macromolecules and nanosized particles including polymer-drug conjugates, liposomes, and micelles are unable to diffuse through the cell membrane into the intracellular compartment. In order to enter the cell, these drug delivery vehicles require an internalization mechanism such as phagocytosis or pinocytosis. Only certain cell types called phagocytic cells (macrophages, neutrophils) can undergo phagocytosis. Particulate materials with diameters of less than 1 mm can be internalized into these cells in membrane-bound vesicles called phagosomes.112 Conversely, pinocytosis is known to be non-cell type specific. This mechanism occurs by invagination of the cell membrane that is similar to the mechanism in phagocytosis. However, the pinocytic vesicles that pinch off from the cell membrane are generally smaller than phagosomes and have diameters ranging from 100 to 200 nm.112 The pinocytosis mechanism can be further categorized into fluid-phase and adsorptive.

Fluid-phase pinocytosis involves the engulfment of soluble materials and extracellular fluid during continual cell membrane ruffling. This is in contrast to adsorptive pinocytosis that involves the association of particulate materials with cell membrane components prior to internalization.113

FIGURE 17.5 Internalization of ligand-conjugated micelles via receptor-mediated endocytosis. (a) The ligand-coupled micelles approach the cancer cells and (b) bind to the cell surface receptors depending on the association constant (ka) and the dissociation constant (kd) of the receptor-ligand complexes. (c) The micelles are then rapidly internalized into the cells via receptor-mediated endocytosis. (d) The internalized receptor and the ligand-conjugated micelles then undergo endosomal sorting in the early endosomes. In most cases, (e) the receptor is recycled to the cell surface, and (f) the micelles enter the late endosome that eventually fuses with the lysosome.

FIGURE 17.5 Internalization of ligand-conjugated micelles via receptor-mediated endocytosis. (a) The ligand-coupled micelles approach the cancer cells and (b) bind to the cell surface receptors depending on the association constant (ka) and the dissociation constant (kd) of the receptor-ligand complexes. (c) The micelles are then rapidly internalized into the cells via receptor-mediated endocytosis. (d) The internalized receptor and the ligand-conjugated micelles then undergo endosomal sorting in the early endosomes. In most cases, (e) the receptor is recycled to the cell surface, and (f) the micelles enter the late endosome that eventually fuses with the lysosome.

For example, micelles that are conjugated with specific ligands can bind to the corresponding receptors that are expressed on the cell surface; as a result, the micelles can be internalized via receptor-mediated endocytosis.78,114,115 More details on receptor-mediated endocytosis are provided in Section 17.6 and Figure 17.5. In most cases, micelles are internalized into the cells through fluid-phase endocytosis.77,116,117

Fluid-phase endocytosis is expected to result in a lower degree of cell uptake in comparison to adsorptive pinocytosis or diffusion of free hydrophobic agents. Differences in the cell uptake of micelle-incorporated agents and non-micelle-incorporated agents have been demonstrated in various studies. For example, using a hydrophobic fluorescent probe as a tracker for micelles, the rate and amount of intracellular fluorescence was found to be much lower than incubation with free probes.104 This has also been confirmed by other studies that relied on confocal microscopy as a qualitative tool.103,105 Rapoport et al. also demonstrated that the incorporation of doxorubicin and ruboxyl into Pluronic® micelles resulted in a decrease in the extent of cell uptake of the agents in ovarian cancer cells.118 In a drug accumulation study at copolymer concentrations above the CMC of the Pluronic® copolymers, the accumulation of the fluorescent probe R123 was substantially decreased in both non-multi- and multi-drug resistant cancer cells. It was suggested that the R123-loaded micelles were transported into the cells via a vesicular route and followed accumulation kinetics different from that of the non-micelle incorporated R123.34

Endocytosis is known to be an energy-, time-, and pH-dependent process.113 Many pharmacological manipulations have been employed in order to demonstrate that micelles are internalized via an endocytotic pathway. For example, a low pH and incubation at 4°C resulted in the reduction of the internalization of micelles.104 Also, pre-treatment of cells with metabolic inhibitors such as sodium azide and 2-deoxyglucose (DOG) that deplete the energy source for membrane ruffling resulted in a decrease in the extent of uptake of radiolabeled-drug loaded-PEG-b-PCL micelles by at least 80%. Another study conducted by Luo et al. reported similar findings, yet in this case, the micelles were tracked using fluorescently labeled-copolymer.106 Furthermore, the pre-treatment of cells with Brefeldin A, known to alter the morphology of endosomes, was also found to reduce the extent of uptake of micelles. This suggests that an endosomal compartment may be involved in the internalization of the micelles that further supports the claim that micelle internalization does proceed by an endocytotic mechanism.104

In order to confirm that micelles have been internalized via the endocytotic pathway, different methods have been developed for the detection of micelles in the relevant organelles. For example, a pH-sensitive fluorescent probe has been used to confirm the presence of the micelles in acidic organelles using flow cytometry.118,119 The subcellular localization of micelles can also be visualized using confocal microscopy by fluorescently labeling the micelles and counterstaining the intracellular organelles.41,105,115 Hydrophobic fluorescent probes physically entrapped in micelles can also be used to track the micelles. The use of these probes to follow the pathway or fate of the micelles requires virtually no or minimal release of the probe during the time frame of the experiments. However, cell culture media often contain protein that can act to accelerate drug release.120 Therefore, the accuracy associated with using physically entrapped probes to track micelles is questionable because of the different subcellular localization patterns of free and micelle-incorporated drugs.41,105 Ideally, fluorescent probes that are chemically conjugated to the copolymers provide the most reliable tool for following the micelles in vitro. Although copolymers also exist in unimeric form, the concentration of unimers in a micelle solution does not exceed the CMC of the copolymer and represents only a minor population.106 Therefore, it is reasonable to assume that the fluorescence detected corresponds to the micelle population provided that the conjugation of the probe to the copolymers is stable.

Internalized micelles have been shown to localize mainly in cytoplasmic compartments, in particular, the accumulation of micelles in acidic organelles (endosomes or lysosomes) has been observed.105,118,119 Rapoport et al. have reported on the intracellular uptake and trafficking of Pluronic® micelles. Their results suggested that after the micelles were internalized in acidic organelles, the non-ionic Pluronic® copolymers induced permeabilization of the membranes, resulting in the release of the encapsulated drugs into the cytosol or into the nucleus via diffusion of the drug.118 The nuclear pore complexes on the nucleus allow for passive diffusion of small molecules that have diameters of less than 9 nm, whereas macromolecules as large as 39 nm can be actively transported into the nucleus.121 Theoretically, micelles that are greater than 9 nm cannot be translocated into the nucleus unless nuclear targeting signals are present on the particles. For example, in cancer cells that have been incubated with tetramethylrhodamine-5-carbonyl azide (TMRCA)-conjugated PEG-b-PCL micelles for 24 h, only cytoplasmic localization of the micelles was observed, and the nuclear compartment was devoid of micelles.105 Similar results were obtained by Shuai et al. with doxorubicin-loaded PEG-b-PCL micelles that have a delayed release profile for doxorubicin from the micelles. The authors demonstrated that following incubation of cells with micelle formulated doxorubicin for 2 and 24 h, the drug was only localized in cytoplasmic compartments, whereas incubation of free drug with cells resulted in nuclear accumulation.41 To date, the cellular internalization pathway and fate of micelles and micelle-encapsulated drugs as a function of time have not been clearly elucidated. However, the in vitro pathway and fate of micelles can have a significant impact on the cytotoxic effects of the formulated drug. The various anti-cancer agents that have been traditionally formulated in micelles have different sites of action within the cell. For example, paclitaxel and doxorubicin are required to bind to microtubules in the cytoplasm and DNA in the nucleus, respectively, in order to exert their cytotoxic effects. Therefore, if micelles could be engineered or designed to achieve a specific subcellular localization that matched the required localization of the drug, the cytotoxicity of the drug could be enhanced.

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