Figure 1.
Rhodamine-based flurone dyes.
Figure 2.
Spectroscopic characteristics of R123 and R110.
1 µM R123 or R110 were prepared in 1% (v/v) MeOH:HBSS. (A) λmax for excitation of each fluorophore was determined using a wavelength absorbance scan (400–600 nm), and (B) λmax for emission determined using a wavelength scan (500–600 nm) with a fixed λex = 505 nm. (C+D) 1 µM R123 or R110 in 1% (v/v) indicated sovent:HBSS were examined with an emission wavelength scan (500–600 nm) with a fixed λex = 505 nm for (C) R123 and (D) R110. (E) Quantum yield for R123 was determined for each solvent combination and plotted against solvent dielectric constant. (F) The impact of commonly used extraction buffers on R123 quantum yield was determined by addition of 1 µM R123 to extraction buffers, followed by an emission wavelength scan (500–600 nm) undertaken with a fixed λex = 505 nm. All data are representative of at least three independent repeats.
Figure 3.
Passive uptake and sequestration of R123.
(A) Critical micelle concentration of R123 was determined by measuring quantum yield (λex = 505 nm, λem = 5251nm) of 0–0.5 µM R123 in 1% (v/v)MeOH:HBSS. (B) MDCKII-ABCB1 cells were incubated with 0–20 µM R123 for 10 minutes and intracellular R123 concentration determined against a standard curve following cell lysis with Triton ×100. (C) MDCKII or Huh7 cells were incubated with 10 µM R123 for 10 minutes and R123 concentration in soluble and insoluble cellular fractions determined against R123 standard curves. (D) Albumin binding was determined through the measurement of 0.1 µM R123 fluorescence following the addition of increasing quantities of 0–15 µM albumin. (E) S9 metabolic fractions were extracted from human liver samples and MDCKII cells and used at a final concentration of and respectively. The rate of conversion of R123 to R110 was determined following addition of 0.1 mg/ml or 1 mg/ml S9 fraction from MDCKII cells or human liver, respectively. Rate of conversion was determined for the indicated range of R123, and fitted using an allosteric sigmoidal model of enzyme kinetics. Data points are the average of three separate repeats ± S.D; where no error bars are observed, they are contained within the limits of the data point.
Figure 4.
Carrier-protein dependent transport of R123 through biological membranes.
(A–D) HEK-293 MSRII cells were singly transduced with BacMam viruses carrying human CDS' for OATP1A2, OATP1B3, OATP2B1 and OATP1B1 and incubated for 48 hours to allow protein expression. Cells were exposed to 0–2 µM R123, in the presence (open circles) or absence (closed circles) of 10 nM specific inhibitor for 120, 300, 420 and 600 seconds at 37°C, and intracellular fluorescence determined: Inhibitors were ketoconazole (OATP1A2), rifamycin (OATP1B1/3) and montelukast (OATP2B1). Data points correspond to the rate of uptake obtained from linear regressions over three time points with duplicate values at each concentration. (E) Naive MDCKII cells (open circles), or MDCKII cells with the CDS for human ABCB1 stably integrated (closed circles) were loaded with varying concentrations of R123 and efflux measured over 7.5 minutes. For all panels, inset images represent Western blot analysis for naïve and transfected cells (top row), plus actin loading control (bottom row), demonstrating high-level expression of the requisite human transporter in each case.
Table 1.
Optimal conditions for BacMam transduction of transporter expression plasmids in HEK293-MSRII cells.