nihms122757.pdf

Methods to Explore Cellular Uptake of Ruthenium Complexes

Cindy A. Puckett

and

Jacqueline K. Barton

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

California 91125

Abstract

The cellular uptake of a series of dipyridophenazine (dppz) complexes of Ru(II) was examined by

flow cytometry. The complexes, owing to their facile synthesis, stability, and luminescence, provide

a route to compare and contrast systematically factors governing cellular entry. Substituting the

ancillary ligands in the dppz complexes of Ru(II) permits variation in the overall complex charge,

size, and hydrophobicity. In HeLa cells, cellular uptake appears to be facilitated by the lipophilic

4,7-diphenyl-1,10-phenanthroline (DIP) ligand. Despite the large size of Ru(DIP)

dppz

(20 Å

diameter), this complex is readily transported inside the cell compared to smaller and more

hydrophilic complexes such as Ru(bpy)

dppz

. Accumulation in the cellular interior is confirmed

by confocal microscopy.

The cellular uptake characteristics of a small molecule are critical to its application as a

therapeutic or diagnostic agent. However, our understanding of the chemical rules governing

uptake is rudimentary.

Although transition metal complexes have increasingly been applied

for biological applications,

–

their uptake properties are even less well developed. Here, we

exploit flow cytometry to provide statistics on the uptake of ruthenium complexes into HeLa

cells. These ruthenium complexes, owing to their facile synthesis, stability, and luminescence,

provide a route to compare and contrast factors governing cellular uptake.

A series of dipyridophenazine (dppz) complexes of Ru(II) was synthesized for systematic

comparison.

–

Substituting the ancillary ligands on the dppz complex permits variation in the

overall complex charge, size, and hydrophobicity (Figure 1). Furthermore since these dppz

Email: jkbarton@caltech.edu.

Supporting Information Available. Flow cytometry for cell nuclei. This material is available free of charge via the Internet at

http://pubs.acs.org.

NIH Public Access

Author Manuscript

J Am Chem Soc

. Author manuscript; available in PMC 2009 September 21.

Published in final edited form as:

J Am Chem Soc

. 2007 January 10; 129(1): 46–47. doi:10.1021/ja0677564.

NIH-PA Author Manuscript

complexes all act as molecular light switches, showing minimal luminescence in aqueous

solution and intense luminescence when bound to DNA or otherwise protected from water,

they provide a sensitive cellular probe (Table 1).

–

HeLa cells were prepared for flow cytometry analysis after incubation with the Ru complexes

at various concentrations and times.

Flow cytometry was performed on a BD FACS Aria

using ~20,000 cells per sample. The ruthenium complexes were excited at 488 nm, with the

emission observed at 600–620 nm. Live cells were distinguished by their low To-Pro-3

emission.

Figure 2 illustrates results of the flow cytometry. Cells not treated with complex exhibit some

background luminescence. Incubation with 10

M Ru(bpy)

dppz

or Ru(phen)

dppz

for 2

h causes only a small change in the luminescence profile. When cells are incubated with 10

M Ru(DIP)

dppz

, however, the luminescence intensity of the cell population increases

dramatically.

Uptake for the different Ru complexes may be compared based upon the mean luminescence

intensity of the cell population (Table 2). Below 1

M, Ru(DIP)

dppz

is taken up appreciably

above background. At higher concentrations, Ru(bpy)

dppz

, Ru(CO

Et-bpy)

dppz

, and

Ru(phen)

dppz

are taken up to some extent, but even at 20

M Ru, little luminescence is

evident for Ru(mcbpy)

dppz.

Washing with buffer reduces luminescence by 20–50%,

suggesting that, while some Ru is non-specifically adhered to the surface or rapidly exported,

the bulk of Ru remains.

That the complexes are actually transported into the cellular interior rather than associating

solely at the membrane surface is evident by confocal microscopy (Figure 3).

For Ru

(DIP)

dppz

, intense luminescence in the interior is apparent within 2 h. For Ru

(phen)

dppz

and Ru(bpy)

dppz

, microscopy experiments also show uptake into the

cellular interior but, consistent with the flow cytometry data, on a slower time scale (

≥

4 h for

phen). For all complexes, greatest luminescence is evident in the cytoplasm, likely associated

with the mitochondria and endoplasmic reticulum based upon costaining experiments with

organelle-specific dyes;

without protection from water through macromolecular binding, Ru

quenching in the cytosol is expected.

Interestingly, significantly less luminescence is apparent in the nucleus. Quantitation by line

plots (Figure 3) does show nuclear uptake but of diminished intensity in the nucleus compared

to other regions.

Flow cytometry experiments on nuclear preparations isolated after

incubation of cells with Ru(DIP)

dppz

provide consistent evidence of Ru uptake (Supporting

Information).

These data establish that the ruthenium complexes are indeed taken up inside HeLa cells. If

the complexes are entering the cell by passive diffusion, one would predict that neutral charge,

smaller size, and greater hydrophobicity should aid uptake. Here, however, cellular uptake

appears to be facilitated by the lipophilic DIP ligand, despite the larger size of the complex. It

is surprising that the large expanse of the DIP complex does not limit its uptake. Also of interest,

changing the overall charge from +2 to neutral does not improve uptake, based upon the low

luminescence results for Ru(mcbpy)

dppz.

There have been few systematic studies on the cellular uptake of transition metal complexes

reported.

–

Our results are in agreement with studies on cisplatin analogues, where the

complexes with the greatest lipophilicity exhibit the highest uptake; note that for the Pt

complexes, all were hydrophilic, with octanol/water partition coefficients of <1.

Importantly,

these data also establish that the Ru complexes are stable to the intracellular environment; no

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degradation in luminescence is evident, as would be expected based upon changes in complex

coordination.

Flow cytometry, traditionally used to examine organic fluorophores, thus provides an

opportunity to examine the cellular uptake of transition metal complexes. Ruthenium analogues

in particular can be readily tested without special instrumentation or complicated synthesis.

Statistics on thousands of cells of varied cell type and using a range of metal complexes can

be generated to provide a powerful complement in the design of metal complexes for biological

application.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

We are grateful to the NIH (GM33309) for their financial support. We also thank the Caltech Flow Cytometry Facility

and the Caltech Biological Imaging Center.

References and Footnotes

1. DeVito, SC. Handbook of Property Estimation Methods for Chemicals: Environmental and Health

Sciences. Boethling, RS.; Mackay, D., editors. CRC Press; Boca Raton, FL: 2000. p. 261-278.

2. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Adv Drug Delivery Rev 2001;46:3–26.

3. Zhang CX, Lippard SJ. Curr Opin Chem Biol 2003;7:481–489. [PubMed: 12941423]

4. Boerner LJK, Zaleski JM. Curr Opin Chem Biol 2005;9:135. [PubMed: 15811797]

5. (a) Brunner J, Barton JK. Biochemistry 2006;45:12295–12302. [PubMed: 17014082] (b) Hart JR,

Glebov O, Ernst RJ, Kirsch IL, Barton JK. Proc Nat Acad Sci 2006;103:15359–15363. [PubMed:

17030786]

6. Dickeson JE, Summers LA. Aust J Chem 1970;23:1023–1027.

7. Ru(phen)

, and Ru(DIP)

were synthesized in analogous fashion to Ru(bpy)

described in

Sullivan BP, Salmon DJ, Meyer TJ. Inorg Chem 1978;17:3334–3341.3341. The dppz ligand was then

added by refluxing in ethanol-water for > 3 h.

8. Ru(CO

Et-bpy)

was synthesized using a modification of Leasure RM, Ou W, Moss JA, Linton

RW, Meyer TJ. Chem Mater 1996;8:264–273.273. 2:1 DME-ethanol was used as the reaction solvent.

The dppz ligand was added by refluxing in EtOH for 24 h. Ru(mcbpy)

dppz was formed by hydrolysis

of the ester.

9. (a) Friedman AE, Chambron JC, Sauvage JP, Turro NJ, Barton JK. J Am Chem Soc 1990;112:4960.

(b) Jenkins Y, Friedman AE, Turro NJ, Barton JK. Biochem 1992;31:10809–16. [PubMed: 1420195]

10. Olofsson J, Onfelt B, Lincoln P. J Phys Chem A 2004;108:4391.

11. Ardhammer M, Lincoln P, Norden B. J Phys Chem B 2001;105:11363–11368.

12. HeLa cells were detached from monolayer culture with EDTA, resuspended in Hank’s Balanced Salt

Solution supplemented with bovine serum albumin fraction 5, and diluted to 1×10

cells/mL. The

ruthenium complexes were added to the cell suspensions at concentrations of 0.5–10

M and

incubated for 2 h at ambient temperature. Dead cells were stained with 1

M To-Pro-3 (Molecular

Probes), which enters cells having compromised membranes.

13. The low luminescence from Ru(mcbpy)

dppz may be in part due to poor nucleic acid binding by the

neutral complex.

14. Confocal microscopy of cells incubated with a Ru dimer has been reported. See Önfelt B, Gostring

L, Lincoln P, Nordén B, Önfelt A. Mutagenesis 2002;17:317–320.320 [PubMed: 12110628]

15. Haugland, RP. Handbook of Fluorescent Probes and Research Products. Vol. 9. Gregory, J.; Spence,

M., editors. Molecular Probes; Eugene, OR: 2002. p. 473-488.p. 496-502.

16. After incubation of HeLa cells for 2 h with 5

M Ru(DIP)

dppz

, line plot quantitation shows an

average of 11% luminescence in the nucleus compared to cytoplasm; for 10

M Ru after 12 h, as in

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Figure 3b, the ratio is ~ 30%. Some contribution to fluorescence in the nucleus could arise from

cellular autofluorescence.

17. The lower luminescence of Ru(mcbpy)

dppz with nucleic acids contributes to its relatively poor

luminescence in cells but cannot fully account for it.

18. Jonas SK, Riley PA. Cell Biochem Function 1991;9:245–253.

19. Kalayda GV, Fakih S, Bertram H, Ludwig T, Oberleithner H, Krebs B, Reedijk J. J Inorg Biochem

2006;100:1332–1338. [PubMed: 16684566]

20. Ghezzi A, Aceto M, Cassino C, Gabano E, Osella D. J Inorg Biochem 2004;98:73–78. [PubMed:

14659635]

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Figure 1.

Dipyridophenazine complexes of Ru(II).

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Figure 2.

Flow cytometry analysis of HeLa cells incubated with 10

M ruthenium complex for 2 h.

Luminescence data were obtained by excitation at 488 nm with emission at 600–620 nm using

a light scatter gate to exclude debris and To-Pro-3 (exciting at 633 nm and observing at 650–

670 nm) to exclude dead cells.

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Figure 3.

Confocal microscopy of HeLa cells with Ru(DIP)

dppz

. (top) Microscopy after incubation

with 5

M Ru for 2 h at 37 °C. Excitation wavelength = 488 nm. (bottom) Intensity profile of

ruthenium luminescence across a HeLa cell after incubation for 12 h with 10

M Ru.

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Table 1

Characteristics of Ru complexes

Ancillary Ligand of

RuL

dppz

Relative emission

intensity in

Relative emission

intensity w/

DNA

Octanol/H

O partition

coefficient (log P)

Diameter (Å)

bpy

1.0

−

2.50

16.2

Et-bpy

1.8

1.1

−

0.76

20.4

mcbpy

1.2

0.6

−

0.43

18.2

phen

1.2

2.3

−

1.48

16.2

DIP

2.7

1.30

20.4

Excited at 488 nm; integrated emission at 600–620 nm. 10

M Ru was used, except for Ru(DIP)

dppz

in Tris buffer, where a lower concentration

was used due to poor solubility; emission values were scaled accordingly.

Luminescence values with DNA were obtained at saturation.

−

salt.

Diameters were estimated using Titan.

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Table 2

Mean Luminescence Intensity of HeLa Cells Incubated with Ruthenium Complex by Flow Cytometry

Ancillary Ligands of RuL

dppz

Conc. (

bpy

Et-bpy

mcbpy

phen

DIP

0.5

n.d.

597

974 (571)

48 (27)

51 (29)

26 (19)

111 (50)

n.d.

Cells were incubated with ruthenium complex for 2 h at ambient temperature. Ruthenium complexes were excited at 488 nm, with emission observed at 600–620 nm. The mean luminescence intensity

of cells not treated with complex is 23. Data are an average of two independent experiments, and data not determined are indicated by n.d.

Samples washed after incubation are shown in parenthesis.

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. Author manuscript; available in PMC 2009 September 21.