P-glycoprotein inhibitors: synthesis and in vitro evaluation of a preactivated thiomer.

The aim of this study was to synthesize the preactivated thiomer poly(acrylic acid)-cyteine-2-mercaptonicotinic acid (PAA-Cys-2MNA) and to evaluate its P-glycoprotein (P-gp) inhibitory properties. The thiomer (PAA-Cys) was synthesized by covalent immobilization of thiol groups on poly(acrylic acid) (PAA) with a molecular mass of 250 kDa followed by immobilization of 2-mercaptonicotinic acid (2MNA) to thiol groups via disulfide bond formation resulting in PAA-Cys-2MNA. P-gp inhibitory effect of this preactivated thiomer was evaluated on Caco-2 cells. Transports of rhodamine 123 at 37 °C with and without verapamil and at 4 °C were performed to evaluate P-gp function of cells. In total, 1571.81 ± 156.18 µmol thiol groups were immobilized per gram of polymer that were in the next step by 99.88% preactivated. The enhancement ratios of Papp calculated from the ratio between Papp of rhodamine 123 in the presence of P-gp inhibitors and Papp of rhodamine 123 alone were 2.36, 2.09, and 1.84-fold in the presence of PAA-Cys-2MNA, PAA-Cys, and PAA, respectively. Because of its pronounced P-gp inhibitory effect, PAA-Cys-2MNA could be considered as promising macromolecular P-gp inhibitor for various drug delivery systems.

Synthesis and characterization of macromolecular rhodamine tethers and their interactions with P-glycoprotein.

Rhodamine dyes are well-known P-glycoprotein (P-gp) substrates that have played an important role in the detection of inhibitors and other substrates of P-gp, as well as in the understanding of P-gp function. Macromolecular conjugates of rhodamines could prove useful as tethers for further probing of P-gp structure and function. Two macromolecular derivatives of rhodamine, methoxypolyethylene glycol-rhodamine6G and methoxypolyethylene glycol-rhodamine123, were synthesized through the 2'-position of rhodamine6G and rhodamine123, thoroughly characterized, and then evaluated by inhibition with verapamil for their ability to interact with P-gp and to act as efflux substrates. To put the results into context, the P-gp interactions of the new conjugates were compared to the commercially available methoxypolyethylene glycol-rhodamineB. FACS analysis confirmed that macromolecular tethers of rhodamine6G, rhodamine123, and rhodamineB were accumulated in P-gp expressing cells 5.2 ± 0.3%, 26.2 ± 4%, and 64.2 ± 6%, respectively, compared to a sensitive cell line that does not overexpress P-gp. Along with confocal imaging, the efflux analysis confirmed that the macromolecular rhodamine tethers remain P-gp substrates. These results open potential avenues for new ways to probe the function of P-gp both in vitro and in vivo.

[¹¹C]Rhodamine-123: synthesis and biodistribution in rodents.

INTRODUCTION: Rhodamine-123 is a known substrate for the efflux transporter, P-glycoprotein (P-gp). We wished to assess whether rhodamine-123 might serve as a useful substrate for developing probes for imaging efflux transporters in vivo with positron emission tomography (PET). For this purpose, we aimed to label rhodamine-123 with carbon-11 (t(1/2)=20.4min) and to study its biodistribution in rodents. METHODS: [¹¹C]Rhodamine-123 was prepared by treating rhodamine-110 (desmethyl-rhodamine-123) with [¹¹C]methyl iodide. The biodistribution of this radiotracer was studied with PET in wild-type mice and rats, in efflux transporter knockout mice, in wild-type rats pretreated with DCPQ (an inhibitor of P-gp) or with cimetidine (an inhibitor of organic cation transporters; OCT), and in P-gp knockout mice pretreated with cimetidine. Unchanged radiotracer in forebrain, plasma and peripheral tissues was also measured ex vivo at 30min after radiotracer administration to wild-type and efflux transporter knockout rodents. RESULTS: [(¹¹C]Rhodamine-123 was obtained in 4.4% decay-corrected radiochemical yield from cyclotron-produced [¹¹C]carbon dioxide. After intravenous administration of [¹¹C]rhodamine-123 to wild-type rodents, PET and ex vivo measurements showed radioactivity uptake was very low in brain, but relatively high in some other organs such as heart, and especially liver and kidney. Inhibition of P-gp increased uptake in brain, heart, kidney and liver, but only by up to twofold. Secretion of radioactivity from kidney was markedly reduced by OCT knockout or pretreatment with cimetidine. CONCLUSIONS: [¹¹C]Rhodamine-123 was unpromising as a PET probe for P-gp function and appears to be a strong substrate of OCT in kidney. Cimetidine appears effective for blocking OCT in kidney in vivo.

Carbon nanotubes as intracellular carriers for multidrug resistant cells studied by capillary electrophoresis-laser-induced fluorescence.

Fluorescently labeled carbon nanotube probes (CNTP) are prepared by derivatizing oxidized (o)-MWNTs with a fluorescein dye. Capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) detection is used to separate and detect CNTP in multidrug-resistant cells (K562A) and the parent cells (K562S). CE-LIF and flow cytometry investigation reveal that the CNTP can traverse the membranes in both cell lines without being pumped out by P-glycoprotein. The CE-LIF method is also useful for quantitative analysis of CNT in single cells, enabling drug delivery and multidrug resistance (MDR) studies. Moreover, toward quantifying the intracellular uptake of oxidized (o)-SWNTs with anchored Rhodamine123 (Rho123), fluorescence-quenching of Rho123 is measured by micellar electrokinetic chromatography coupled with LIF detection. Enhanced uptake of Rho123 in multidrug-resistant leukemia cells can be achieved with the aid of the o-SWNTs carriers. Besides being able to overcome MDR, o-SWNTs are shown to be excellent intracellular carriers possessing large adsorption capacity and prolonged release ability. Finally, it is demonstrated that o-SWNTs are safe for biological applications at concentrations of up to 40 microg/mL.

A specific tumor-targeting magnetofluorescent nanoprobe for dual-modality molecular imaging.

Poly(acrylic acid) was decorated onto Fe(3)O(4) resulting in a highly water-soluble superparamagnetic iron oxide. The Poly(acrylic acid) iron oxide (PAAIO) complexes possess specific magnetic properties in the presence of an external magnetic field and are attractive contrast agents for magnetic resonance imaging (MRI). The free carboxylic groups of PAAIO exposed on the surface allow for covalent attachment of a fluorescent dye, Rhodamine 123 (Rh123) to form PAAIO-Rh123, which permits applications in fluorescence imaging. PAAIO-Rh123 is therefore a dual-modality molecular probe. In order to endow specific properties to compounds that target cancer cells and to prevent recognition by the reticuloendothelial system (RES), folic acid-linked poly(ethylene glycol) (FA-PEG) was further conjugated onto PAAIO-Rh123. The amounts of Rh123 and FA-PEG on the modified iron oxides were quantitatively determined by elemental analysis. The iron content was determined by inductively coupled plasma-optical emission spectrometer (ICP-OES). The particle diameters were characterized by dynamic light scattering (DLS) and transmission electron microscope (TEM). Superparamagnetism was confirmed by the superconducting quantum interference device (SQUID) magnetometer. The cellular internalization efficacy of the modified iron oxides was realized in folate-overexpressed FR(+) and folate-deficient FR(-) KB cells by flow cytometry and confocal laser scanning microscopy (CLSM). The quantitative amount of iron internalized into different harvested KB cells was measured by ICP-OES. The T(2)-weighted MR images were tested in FR(+) KB cells.