Characterization of Rhodamine-123 as a Tracer Dye for Use In In vitro Drug Transport Assays

Fluorescent tracer dyes represent an important class of sub-cellular probes and allow the examination of cellular processes in real-time with minimal impact upon these processes. Such tracer dyes are becoming increasingly used for the examination of membrane transport processes, as they are easy-to-use, cost effective probe substrates for a number of membrane protein transporters. Rhodamine 123, a member of the rhodamine family of flurone dyes, has been used to examine membrane transport by the ABCB1 gene product, MDR1. MDR1 is viewed as the archetypal drug transport protein, and is able to efflux a large number of clinically relevant drugs. In addition, ectopic activity of MDR1 has been associated with the development of multiple drug resistance phenotype, which results in a poor patient response to therapeutic intervention. It is thus important to be able to examine the potential for novel compounds to be MDR1 substrates. Given the increasing use rhodamine 123 as a tracer dye for MDR1, a full characterisation of its spectral properties in a range of in vitro assay-relevant media is warranted. Herein, we determine λmax for excitation and emission or rhodamine 123 and its metabolite rhodamine 110 in commonly used solvents and extraction buffers, demonstrating that fluorescence is highly dependent on the chemical environment: Optimal parameters are 1% (v/v) methanol in HBSS, with λex = 505 nm, λem = 525 nm. We characterise the uptake of rhodamine 123 into cells, via both passive and active processes, and demonstrate that this occurs primarily through OATP1A2-mediated facilitated transport at concentrations below 2 µM, and via micelle-mediated passive diffusion above this. Finally, we quantify the intracellular sequestration and metabolism of rhodamine 123, demonstrating that these are both cell line-dependent factors that may influence the interpretation of transport assays

Passive uptake and sequestration of R123.

<p>(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.</p

Spectroscopic characteristics of R123 and R110.

<p>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.</p

Optimal conditions for BacMam transduction of transporter expression plasmids in HEK293-MSRII cells.

<p>Pfu = plaque forming units; MOI = multiplicity of Infection.</p