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Effect of amphiphilic diblock copolymers on P-glycoprotein substrate permeability in Caco-2 cells Zastre, Jason


A series of short block length methoxypoly(ethylene g\yco\)-blockpoly( caprolactone) (MePEG-b-PCL) diblock copolymers were synthesized, characterized and evaluated for enhancing the permeability of P-gp substrates in an intestinal epithelial cell line, caco-2. Altering MePEG:caprolactone feed weight ratios produced diblocks composed of varying PCL lengths, with MePEG of MW 550, 750 or 2000. The diblocks formed micelles above the critical micelle concentration (CMC) values and hydrophobicity, solubility, CMC, and micelle size, were dependent on the block lengths of the diblock copolymers. Caco-2 cellular accumulation studies with two homologous P-glycoprotein substrates, rhodamine 123 (R-123) and rhodamine 6G (R-6G), showed that accumulation of the relatively hydrophilic P-gp substrate, R-123, was enhanced at high concentrations of MePEG-b-PCL diblock copolymers above their CMC with little activity below the CMC. Whereas, cellular accumulation with the relatively hydrophobic substrate, R-6G, was maximally enhanced over a wide range of diblock concentrations, lower or close to the CMC and which also corresponded to an 8-25 fold reduction in diblock concentration compared to R-123. Diblocks with intermediate to high HLB values were more effective at enhancing R- 123 accumulation, while all diblocks were shown to enhance R-6G accumulation. Similar accumulation enhancement profiles with MePEG-b-PCL were observed with two additional P-gp substrates, doxorubicin and paclitaxel, which have large differences in their relative hydrophobicities. At high diblock copolymer concentrations, R-123 and R-6G accumulation decreased and was likely due to substantial partitioning of R-123 and R-6G into micelles, reducing the free fraction available for cellular uptake. A substantially different rate of substrate accumulation with MePEG-b-PCL was observed depending upon the hydrophobicity of the P-gp substrate. MePEG-b-PCL diblocks increased the rate and extent of R-123 accumulation, but not the rate of accumulation of R-6G. The difference in MePEG-b-PCL diblock composition and concentrations required to enhance the accumulation of P-gp substrates with different relative hydrophobicities suggests that additional pathways may be involved besides a reduction of P-gp mediated efflux. To determine the cellular uptake pathways contributing to enhanced caco-2 cellular accumulation of P-gp substrates by MePEG-b-PCL block copolymers, the effects of endocytosis inhibitors, ATP depletion conditions, and directional transepithelial flux experiments were performed. For the hydrophilic R-123, MePEG₁₇-b-PCL₅ enhanced the cellular accumulation of R-123 at high concentrations of diblock above the CMC, which did not appear to involve endocytosis of micellized R-123. This suggests that MePEG₁₇-b-PCL₅ micelles may provide a 'depot' for free unimer to interact with the cell membrane and contribute either to enhanced passive transmembrane diffusion of R-123 through membrane permeability changes, or inhibition of P-gp mediated efflux, or both. In the secretory direction, diblock was capable of reducing the efflux of both R-123 and R-6G. However, MePEG₁₇-b-PCL₅ greatly enhanced the cellular accumulation of R-123 in the transepithelial directional flux studies in the absence of an increase in the absorptive flux. It is proposed that the basolateral membrane permeability of R-123 may limit the absorptive flux. In contrast, MePEG₁₇-b-PCL₅ was able to enhance the absorptive flux of the hydrophobic R-6G. ATP depletion studies demonstrated that MePEG₁₇-b-PCL₅ increased the accumulation of R-123 possibly through a membrane permeabilization effect. Erythrocyte hemolysis studies also provided evidence that MePEG₁₇-b-PCL₅ caused membrane perturbation effects, which could result in enhanced transmembrane diffusion of R-123.

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