nihms91079.pdf

Mechanism of cellular uptake of a ruthenium polypyridyl

complex

†

Cindy A. Puckett

and

Jacqueline K. Barton

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA

91125

Abstract

Transition metal complexes provide a promising avenue for the design of therapeutic and diagnostic

agents, but the limited understanding of their cellular uptake is a roadblock to their effective

application. Here, we examine the mechanism of cellular entry of a luminescent ruthenium(II)

polypyridyl complex, Ru(DIP)

dppz

(where DIP = 4,7-diphenyl-1,10-phenanthroline and dppz =

dipyridophenazine), into HeLa cells, with the extent of uptake measured by flow cytometry. No

diminution of cellular uptake is observed under metabolic inhibition with deoxyglucose and

oligomycin, indicating an energy-independent mode of entry. The presence of organic cation

transporter inhibitors also does not significantly alter uptake. However, the cellular internalization

of Ru(DIP)

dppz

is sensitive to the membrane potential. Uptake decreases when cells are

depolarized with high potassium buffer and increases when cells are hyperpolarized with

valinomycin. These results support passive diffusion of Ru(DIP)

dppz

into the cell.

Transition metal complexes have tremendous potential as diagnostic and therapeutic agents.

They can be exploited for their modularity, reactivity, imaging capabilities, redox chemistry,

and their precisely defined three-dimensional structure. An increasing number of biological

applications have been explored (

1–3

). Complexes that are currently in clinical use include the

platinum anticancer drugs, radiodiagnostic agents containing

99m

Tc, and gadolinium(III)

magnetic resonance imaging contrast agents.

In order to design new metal-based drugs more rationally, an understanding of the physiological

processing of metal complexes is required. Though cellular uptake is critical to the success of

a drug or probe, few mechanistic details are known regarding metal complex uptake. Different

entry mechanisms may be preferred depending on the application, as the mode of entry affects

cell-type specificity, the rate of internalization, and the fate of the compound once inside the

cell. For example, entry by diffusion affords broad cell-type specificity, a great advantage in

the use of fluorescent probes for live cell imaging. Conversely, medicinal chemists may seek

to deliver drugs to target organs, taking advantage of tissue-specific transporters (

4) or receptors

(5). For each mode of entry, there are also drawbacks. With protein-mediated transport, the

degree of modification of the molecule is limited because transport relies on recognition. With

endocytosis, molecules are often trapped in endosomes and face degradation by lysosomal

enzymes.

Ruthenium(II) polypyridyl complexes are useful for studying cellular uptake due to their facile

synthesis, stability in aqueous solution, and luminescence. Using confocal microscopy and

flow cytometry, we have examined the uptake of a series of dipyridophenazine (dppz)

†

Financial support for this work from the National Institutes of Health (Grant GM33309 to J.K.B.) is acknowledged.

*To whom correspondence should be addressed. Email: jkbarton@caltech.edu. Telephone: (626) 395-6075. Fax: (626) 577-4976.

NIH Public Access

Author Manuscript

Biochemistry

. Author manuscript; available in PMC 2009 November 11.

Published in final edited form as:

Biochemistry

. 2008 November 11; 47(45): 11711–11716. doi:10.1021/bi800856t.

NIH-PA Author Manuscript

complexes of ruthenium (

6). Despite its larger size, the lipophilic Ru(DIP)

dppz

, (where DIP

= 4,7-diphenyl-1,10-phenanthroline), illustrated in Figure 1, accumulates in cells more quickly

than Ru(bpy)

dppz

(where bpy = 2,2’-bipyridine) and Ru(phen)

dppz

(where phen = 1,10-

phenanthroline). Ru(DIP)

dppz

enters cells within an hour at micromolar concentrations,

providing a reasonable time window for uptake experiments. Details of the cellular uptake

mechanism of Ru(DIP)

dppz

can then be applied to understanding uptake characteristics of

other structurally similar cationic metal complexes.

The biological activity of ruthenium complexes was first examined by Francis Dwyer in the

1950s, where a full family of tris(polypyridyl) complexes was shown to have bacteriostatic

and anti-viral activities (7). More recently, many ruthenium complexes have been tested for

therapeutic potential (8), and two ruthenium anticancer drugs (NAMI-A and KP1019) have

reached clinical trials (

9,10

). Cellular uptake of some ruthenium(III) complexes appears to be

mediated by the iron transport protein transferrin. KP1019 (indazolium

trans

-[tetrachlorobis

-indazole)ruthenate(III)]) binds transferrin with displacement of a chloride ligand, and is

transported into cells with transferrin by receptor-mediated endocytosis (5). Ruthenium

complexes lacking a labile ligand such as chloride are unlikely to be able to enter cells in this

manner and their mechanism of entry has not been established.

Such coordinatively saturated tris(chelate) ruthenium complexes furthermore serve as close

fluorescent analogs of rhodium complexes that we have explored as potential chemotherapeutic

agents (11). These lipophilic, cationic rhodium complexes target single base mismatches in

DNA and selectively inhibit cellular proliferation in mismatch repair-deficient cell lines (3).

The main routes into a cell are endocytosis, active transport, facilitated diffusion, and passive

diffusion. Due to its lipophilicity and positive charge, Ru(DIP)

dppz

likely traverses the

membrane in response to the membrane potential, similar to other lipophilic cations such as

tetraphenylphosphonium and rhodamine 123 (

12,13

). Here, we use chemical tools to elucidate

the cellular uptake mechanism of Ru(DIP)

dppz

, with the degree of uptake analyzed by flow

cytometry.

EXPERIMENTAL PROCEDURES

Materials

Cell culture reagents, transferrin-AlexaFluor488 conjugate, and To-Pro-3 were purchased from

Invitrogen. Oligomycin, deoxyglucose, and the cation transporter inhibitors were obtained

from Aldrich. Valinomycin was purchased from CalbioChem.

Synthesis of Ru complexes

Ru(DIP)

dppz

and Ru(phen)

dppz

were synthesized as described previously (

6). Briefly,

Ru(DIP)

and Ru(phen)

were synthesized in an analogous fashion to Ru(bpy)

(14). The dipyridophenazine (dppz) ligand was synthesized as previously described (15) and

added to RuL

by refluxing in ethanol-water for > 3 h to make Ru(DIP)

dppz

and Ru

(phen)

dppz

. The ethanol was removed by rotary evaporation, resulting in precipitation of

[Ru(DIP)

dppz]Cl

, which was collected by filtration. Ru(phen)

dppz

was precipitated as

the hexafluorophosphate salt, then returned to the chloride salt by Sephadex DEAE anion

exchange column. The Ru complexes utilized are racemic mixtures of the two enantiomers.

Cell culture

HeLa cells (ATCC, CCL2) were maintained in minimal essential medium alpha with 10% fetal

bovine serum and 1% penicillin-streptomycin (100×).

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Inductively Coupled Plasma Mass Spectrometric (ICP-MS) detection of Ru

HeLa cells were grown to ~30% confluency in 75 cm

flasks and incubated with 5 μM Ru

(DIP)

dppz

for 1 h at 37 °C in either media with serum or media without serum. The solution

from the serum-free incubation was saved for circular dichroism measurements (described

below). The cells were rinsed with PBS, detached with trypsin, and counted The cells were

isolated by centrifugation and digested in concentrated nitric acid for 1.5 h at 60 °C. The

solution was diluted with Millipore water to 2.0 mL for the incubation with serum and 20.0

mL for the serum-free incubation, adding nitric acid to give 2%. The Ru content was measured

using a Hewlett Packard 4500 ICP-MS. Data are reported as the mean ± the standard deviation

(n = 3).

Assay of enantiomeric preference in Ru uptake

HeLa cells were grown to ~30% confluency in 75 cm

flasks and incubated with 5 μM Ru

(DIP)

dppz

for 1 h at 37 °C in media without serum and phenol red. After incubation, the

media was retrieved from the flask. The Ru complex was purified from the media using a

Waters C18 Sep-Pak. The solution was loaded onto a 1 g Sep-Pak equilibrated with water. The

Sep-Pak was then rinsed with 150 mL water and 15 mL 20/80 CH

CN/H

O with 0.1% TFA.

The Ru complex was eluted with 90/10 CH

CN/H

O with 0.1% TFA and lyophilized. For

circular dichroism (CD) measurements, this isolated complex was dissolved in CH

CN to give

8 μM complex. CD spectra were recorded on an AVIV 62 CD Spectrometer.

Metabolic inhibition

HeLa cells were detached from culture and treated with either 50 mM 2-deoxy-

-glucose and

5 μM oligomycin in PBS (to inhibit cellular metabolism) or 5 mM glucose in PBS for 1 h at

37 °C. Both solutions also contained 2.5 mg/mL bovine serum albumin fraction V (BSAV).

The cells were then rinsed and suspended in either PBS with BSAV for the inhibition cells, or

PBS with BSAV and 5 mM glucose for the control cells. The cells were incubated with 10 mg/

L transferrin- AlexaFluor488 or 5 μM Ru(DIP)

dppz

for 1 h at 37 °C. Following incubation,

the transferrin-treated cells were rinsed with PBS and trypsinized to cleave the transferring

receptors from the cell surface (

16). The Ru-treated cells were rinsed. The cells were analyzed

by flow cytometry. Ru-treated cells were not trypsinized following incubation, as rinsed cells

and trypsinized cells gave the same mean luminescence in a control experiment: 282 ± 18 for

the rinsed cells and 284 ± 23 for the trypsinized cells, following a 1 h incubation at 37 °C with

5 μM Ru(DIP)

dppz

Temperature dependence of uptake

HeLa cells were detached from culture and washed with Hanks’ balanced salt solution (HBSS)

supplemented with 2.5 mg/mL BSAV. The cells were incubated with 5 μM Ru(DIP)

dppz

or Ru(phen)

dppz

for 2 h, or 10 mg/L transferrin-Alexa488 for 1 h, in HBSS with BSAV at

4 °C, ambient temperature (20–23 °C), or 37 °C. Transferrin-treated cells were trypsinized

following incubation. The amount of uptake was analyzed by flow cytometry.

Cation transporter inhibition

HeLa cells were detached from culture and washed with buffer (HBSS supplemented with

BSAV), then pre-treated for 20 min with either 1 mM cation transport inhibitor or buffer only.

The cells were then incubated with 5 μM Ru(DIP)

dppz

for 1 h at ambient temperature. The

cells were rinsed with buffer and Ru uptake was analyzed by flow cytometry.

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NIH-PA Author Manuscript

Modulation of membrane potential

HeLa cells were detached from culture and washed three times with either HBSS (containing

5.8 mM K

) or high K

-HBSS (containing 170 mM K

), both supplemented with 2.5 mg/mL

BSAV. Some of the cells in HBSS were pre-treated with 50 μM valinomycin for 30 min at 37

°C. The cells were incubated with 2 μM Ru(DIP)

dppz

for 1 h at 37 °C in one of the following

solutions: HBSS, HBSS with valinomycin (to hyperpolarize the cells), or high K

-HBSS (to

depolarize the cells). The solutions also contained BSAV. After incubation, the cells were

rinsed and the extent of uptake was analyzed by flow cytometry.

Flow cytometry

Cells were detached from culture with EDTA (0.48 mM in PBS) and incubated at 1×10

cells/

mL with Ru complex (added from a concentrated DMSO stock) under conditions described

above, then placed on ice. To-Pro-3 was added at 1 μM immediately prior to flow cytometry

analysis to stain dead cells. The fluorescence of ~20,000 cells was measured using a BD FACS

Aria, exciting with the 488 nm laser for Ru and transferrin-AlexaFluor488, and with the 633

nm laser for To-Pro-3. Emission was observed at 600–620 nm for Ru, 515–545 for

AlexaFluor488, and 650–670 nm for To-Pro-3. Cells exhibiting To-Pro-3 fluorescence were

excluded from the data analysis. Fluorescence data is reported as the mean ± the standard

deviation (n = 3).

RESULTS

Strategy to measure uptake

The dipyridophenazine (dppz) complexes of ruthenium serve as light switches for non-aqueous

environments, luminescing only when bound to the hydrophobic regions of membranes,

nucleic acids, and other macromolecules (17,18). If the Ru complex decomposes or ligand

substitution occurs, the resulting complex would no longer luminesce. Additionally the ligands

themselves are not luminescent. Accordingly, the characteristic luminescence indicates that

the complex one inside the cell remains intact. We can use the luminescence of these ruthenium

complexes to track their cellular uptake in both confocal microscopy and flow cytometry

experiments. Confocal imaging confirms that Ru(DIP)

dppz

accumulates inside the cell

rather than associating solely at the membrane surface, as seen in Figure 1 and previously

(6). Ruthenium luminescence is observed throughout the cytoplasm, though mostly excluded

from the nucleus. Flow cytometry, on the other hand, enables the rapid measurement of

ruthenium luminescence intensity for multiple cell populations. Using primarily flow

cytometry, we can then explore the cellular uptake mechanism of Ru(DIP)

dppz

comparing the ruthenium luminescence following different incubation conditions.

ICP-MS measurement of Ru uptake

Inductively coupled plasma mass spectrometry (ICP-MS) measurements were performed to

quantify the amount of Ru taken up by the cell. HeLa cells were treated with 5 μM Ru

(DIP)

dppz

for 1 h at 37 °C in media with or without serum. ICP-MS measurements give

26.4 ± 6.3 amol of Ru per cell for the incubation in media with serum, and 677 ± 73 amol per

cell for the serum-free incubation. Assuming an average cell volume of 1.7 pL (19) and that

the complex is evenly distributed throughout this volume, the Ru concentration in the cell is

approximately 16 μM for incubation with serum and 398 μM for the serum-free incubation.

These results indicate substantial concentration of the complex within the cell, with serum

acting to attenuate the effective free ruthenium complex available in solution.

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Enantiomeric preference in uptake

As racemic Ru(DIP)

dppz

was used for uptake experiments, we considered whether HeLa

cells preferentially take in one enantiomer over the other. To test for enantioselectivity

associated with uptake, we assayed for enantiomeric enrichment in the supernatant. Following

incubation of Ru(DIP)

dppz

with HeLa cells in serum-free media, the Ru complex was

recovered from the media and the circular dichroism (CD) was examined. Serum-free media

was used for the incubation to remove any potential chiral bias created from interaction of the

complex with serum proteins. ICP-MS determination of the Ru content of the cells confirms

that a significant amount of Ru (~3% of the total) was taken in by the cells. Given this depletion,

we estimate that an enantiomeric preference in uptake of 2.5:1 or greater would be detectable

above instrument noise. However, the CD spectra of Ru(DIP)

dppz

after incubation with

cells is within the level of the noise. This absence in optical activity indicates that any

enantiomeric preference must be below this limit.

Energy-dependent uptake mechanisms

Certain cellular uptake routes are energy-dependent, such as endocytosis (20) and active

protein transport. These pathways are hindered when cells are incubated at low temperature (4

°C instead of 37 °C) or in ATP-depleted environments (e.g. from metabolic poisons). To

determine whether Ru(DIP)

dppz

enters cells by an energy-dependent process, the complex

was incubated with HeLa cells after ATP depletion by deoxyglucose (a glucose analog that

inhibits glycolysis) and oligomycin (an inhibitor of oxidative phosphorylation) (21,22). As

transferrin is internalized by clathrin-mediated endocytosis, fluorescently (AlexaFluor488)

labeled transferrin was used as a positive control. The cellular uptake of Ru(DIP)

dppz

remains essentially unchanged when cells are under metabolic inhibition, with a mean

luminescence of 659 ± 12 compared to 675 ± 10 for the cells not treated with deoxyglucose

and oligomycin (Figure 2). Thus Ru(DIP)

dppz

appears to enter cells by an energy-

independent process. In contrast, metabolic inhibition dramatically reduces the endocytosis of

transferrin, demonstrated by the large decrease in the mean fluorescence from 3034 ± 52 to

210 ± 5.

The temperature dependence of Ru(DIP)

dppz

uptake was also explored. HeLa cells were

incubated with the complex at 4 °C, ambient temperature (20–23 °C), and 37 °C (Figure 3).

The mean Ru luminescence increases with incubation temperature, from 589 ± 17 at 4 °C to

826 ± 71 at 37 °C. Given the lipophilicity of Ru(DIP)

dppz

, the increase in cellular uptake

with temperature could also be the result of improved solubility in buffer at higher

temperatures. To test this hypothesis, the temperature dependence of uptake was studied for a

similar but more soluble complex, Ru(phen)

dppz

. In this case, the mean luminescence

remains roughly the same at the different incubation temperatures, 133 ± 10 at 4 °C and 141

± 3 at 37 °C. Transferrin internalization was significantly more sensitive to temperature, with

the mean fluorescence increasing from 480 ± 7 at 4 °C to 4071 ± 167 at 37 °C.

Effect of organic cation transporter inhibitors

Organisms use polyspecific organic cation transporters (OCTs) for the distribution of

endogenous organic cations and the absorption, distribution, and elimination of cationic drugs

and toxins (23). These transporters facilitate the diffusion of structurally diverse compounds.

OCT substrates are typically organic cations and weak bases, though some neutral compounds

and anions are also transported. Expression of the OCTs varies by tissue type. The carnitine

and cation transporter OCTN2 has the most widespread tissue distribution among the OCTs,

and its expression has been detected in some cancer cell lines, including HeLa (

24). Numerous

compounds that inhibit OCT-mediated transport have been identified. Procainamide inhibits

across the OCT family (including OCT1–3, OCTN1, and OCTN2). Tetraethylammonium ion

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is translocated by most of the OCTs, while other

-tetraalkylammonium salts inhibit OCT1

and OCT2, and for some OCTN1 (25).

To investigate whether Ru(DIP)

dppz

crosses the membrane using an organic cation

transporter, uptake of Ru(DIP)

dppz

in the presence of OCT inhibitors was analyzed. HeLa

cells were incubated with 1 mM inhibitor for 20 min before addition of Ru(DIP)

dppz

for 1

h. For OCTs, the IC

’s of the inhibitors used are generally less than 1 mM. Procainamide has

an IC

of < 3 mM and cimetidine <2 mM for OCTN2. As shown in Figure 4, the extent of

uptake was analyzed by flow cytometry. Significantly, the

-tetraalkylammonium salts and

procainamide do not appreciably alter the cellular uptake of Ru(DIP)

dppz

. Within error,

the cells have a similar mean luminescence intensity. The complex does not appear to use an

organic cation transporter for entry into HeLa cells.

Effect of membrane potential

The plasma membranes of viable cells exhibit a membrane potential (

−

50 to

−

70 mV), with

the inside of the cell negative with respect to the outside (26). As Ru(DIP)

dppz

carries a

positive charge, uptake may be driven by the potential difference across the cell membrane.

The membrane potential in animal cells depends mainly on the K

concentration gradient.

Here, the potential was reduced to close to zero by incubating the cells in buffer with potassium

concentration equivalent to that found intracellularly (~170 mM) (27). This high potassium

buffer (K

-HBSS) was created by replacing sodium salts with equimolar potassium salts, while

hyperpolarization of the membrane was achieved by adding valinomycin to low potassium

buffer (HBSS) (28). Valinomycin is a cyclic peptide that selectively shuttles potassium ions

across the membrane down the electrochemical potassium ion gradient, and it increases

themembrane potential by exporting potassium from the cell.

Ru(DIP)

dppz

was incubated with HeLa cells for 1 h in either HBSS, HBSS with

valinomycin (hyperpolarizes), or K

-HBSS (depolarizes). The amount of Ru complex uptake

was analyzed by flow cytometry (Figure 5). Depolarization of the plasma membrane reduces

the uptake of Ru(DIP)

dppz

, decreasing the mean luminescence of the cells from 232 ± 5 to

154 ± 1. Conversely, hyperpolarization of the membrane with valinomycin clearly promotes

Ru complex uptake, increasing the mean luminescence to 428 ± 13. These results indicate that

the positively charged complex is being driven inside the cells at least in part by the membrane

potential.

DISCUSSION

Cellular uptake of small molecules can occur through energy-dependent (endocytosis, active

transport) and energy-independent (facilitated diffusion, passive diffusion) processes.

Metabolic inhibitors deplete the cell of energy, resulting in diminished uptake of molecules

entering by endocytosis and active transport. HeLa cells treated with the metabolic inhibitors

deoxyglucose and oligomycin show greatly reduced uptake (over 10-fold) of fluorescently-

labeled transferrin, which enters cells by endocytosis. On the other hand, deoxyglucose and

oligomycin treatment cause no reduction of Ru(DIP)

dppz

cellular uptake, suggesting an

energy-independent mode of entry. In fact, the midpoint of the luminescence intensity profile

increases slightly in cells facing metabolic inhibition, though the mean fluorescence remains

approximately the same. If Ru(DIP)

dppz

is actively exported, this efflux would be slowed

under energy depletion and could explain the small increase. In contrast with the metabolic

inhibition data, Ru(DIP)

dppz

uptake decreases slightly with lower temperature (4 °C versus

37 °C), consistent with energy-dependent transport. The difference is not as dramatic as for

transferrin, however, whose fluorescence changes 8-fold. Given that Ru(DIP)

dppz

is poorly

soluble in buffer, this change may be due to improved solubility with temperature. Accordingly,

a similar but more soluble complex, Ru(phen)

dppz

, shows constant uptake at different

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temperatures. Low temperature also increases membrane viscosity, via decreased membrane

fluidity, which can impair diffusion through the membrane.

The possible role of an organic cation transporter (OCT) in translocation of Ru(DIP)

dppz

across the membrane was also explored. These transporters facilitate the diffusion of

endogenous organic cations as well as a variety of drugs and toxins. Ru(DIP)

dppz

uptake

is not significantly altered in cells co-incubated with OCT inhibitors, indicating that the

complex is likely not an OCT substrate. This result is consistent with the large size of Ru

(DIP)

dppz

(~20 Å diameter) compared to known OCT substrates.

The cellular uptake of Ru(DIP)

dppz

is, however, influenced by the membrane potential.

Consistent with diffusion of a positively charged molecule, uptake increases at higher potential

and decreases at lower potential. That severe metabolic impairment does not discourage

complex uptake is also consistent with diffusion. As both passage through a channel or passive

carrier and diffusion directly through the lipid bilayer are energy-independent and respond to

changes in the membrane potential, other factors must be considered to distinguish between

these two mechanisms. The ability of Ru(DIP)

dppz

to enter the cell despite cation

transporter inhibition, its larger size than typical transporter substrates, and its relatively slow

rate of cellular accumulation implicate passive diffusion as the mechanism of entry.

Passive diffusion is less cell-type specific, allows greater freedom for modification of the

complex than transport via membrane proteins, and does not lead to entrapment in endosomes,

as often occurs with endocytosis. As a result, this mechanism of passive diffusion may portend

the broad applicability of metal complexes in different cell types for different intracellular

functions. Certainly these results provide some basis for considering the biological activities

that have already been identified for cationic transition metal complexes (3,7). Significantly,

knowledge of the mechanism of cellular uptake of this ruthenium(II) polypyridyl complex can

now be applied to the design of structurally similar metal complexes for therapeutic and

diagnostic use.

Abbreviations

DIP, 4,7-diphenyl-1,10-phenanthroline; dppz, dipyrido[3,2-

:2’,3’-

]phenazine; phen, 1,10-

phenanthroline; bpy, 2,2’-bipyridine; HBSS, Hanks’ balanced salt solution; PBS, phosphate-

buffered saline; BSAV, bovine serum albumin fraction V; OCT, organic cation transporter;

ICP-MS, inductively coupled plasma mass spectrometry.

ACKNOWLEDGEMENT

We are grateful to the Caltech Flow Cytometry Facility for expert assistance and facilities.

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Puckett and Barton

Page 8

Biochemistry

. Author manuscript; available in PMC 2009 November 11.

NIH-PA Author Manuscript

Figure 1.

A luminescent ruthenium probe used to examine metal complex uptake. Top: Chemical

structure of Ru(DIP)

dppz

. Bottom: HeLa cells incubated with 5 μM Ru(DIP)

dppz

for 4

h, imaged by confocal microscopy. Note that the cytoplasm is extensively stained with the Ru

complex. Scale bar is 10 μm.

Puckett and Barton

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Biochemistry

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NIH-PA Author Manuscript

Figure 2.

Flow cytometry measuring Ru incorporation used to examine the effect of metabolic inhibition

on Ru(DIP)

dppz

cellular uptake. HeLa cells were pretreated for 1 h with 50 mM

deoxyglucose (dGlc) and 5 μM oligomycin, then rinsed and incubated with 5 μM Ru

(DIP)

dppz

or 10 mg/L transferrin-AlexaFluor488 (Tf-Alexa488) for 1 h. Cells treated with

inhibitors (red) are compared to control cells (blue). Top: Ru(DIP)

dppz

uptake. Bottom:

transferrin-AlexaFluor488 uptake.

Puckett and Barton

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Biochemistry

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Figure 3.

Effect of incubation temperature on Ru(DIP)

dppz

cellular uptake measured by flow

cytometry. HeLa cells were incubated with 5 μM Ru(DIP)

dppz

for 2 h at 4 °C, ambient

temperature (20–23 °C), or 37 °C.

Puckett and Barton

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Biochemistry

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NIH-PA Author Manuscript

Figure 4.

Effect of organic cation transporter inhibitors on Ru(DIP)

dppz

cellular uptake measured by

flow cytometry. HeLa cells were pretreated with 1 Mm inhibitor for 20 min, then 5 μM Ru

(DIP)

dppz

was added for 1 h. Each data point is the mean ± the standard deviation of three

samples.

Puckett and Barton

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Biochemistry

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NIH-PA Author Manuscript

Figure 5.

Effect of modulating the plasma membrane potential on Ru(DIP)

dppz

cellular uptake

determined by flow cytometry. HeLa cells were incubated with 2 μM Ru(DIP)

dppz

Hanks’ balanced salt solution (HBSS), HBSS with valinomycin (hyperpolarizes), or K

-HBSS

(depolarizes).

Puckett and Barton

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Biochemistry

. Author manuscript; available in PMC 2009 November 11.

NIH-PA Author Manuscript