Methods to explore cellular uptake of ruthenium complexes

Article abstract of DOI:10.1021/ja0677564

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)₂dppz²⁺ (20 A diameter), this complex is readily transported inside the cell compared to smaller and more hydrophilic complexes, such as Ru(bpy)₂dppz²⁺. Accumulation in the cellular interior is confirmed by confocal microscopy. Copyright

Full text of DOI:10.1021/ja0677564

Published on Web 12/15/2006
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
Received October 30, 2006; E-mail: jkbarton@caltech.edu
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.1,2 Although transition metal complexes have increas-
ingly been applied for biological applications,3-5 their uptake
properties are even less well developed. Here, we exploit flow
cytometry to provide statistics on the uptake of ruthenium com-
plexes 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) were
synthesized for systematic comparison.6-8 Substituting the ancillary
Figure 1. Dipyridophenazine complexes of Ru(II).
ligands on the dppz complex permits variation in the overall
complex charge, size, and hydrophobicity (Figure 1).
Table 1. Characteristics of Ru Complexes
ancillary
ligand of
RuL2dppz
relative
emission intensity
in CH3CNa
relative
emission intensity
w/DNAa,b
octanol/H2O
partition coefficient
(log P)c
Furthermore, since these dppz 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).9-11
HeLa cells were prepared for flow cytometry analysis after
incubation with the Ru complexes at various concentrations and
times.12 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 µM Ru(bpy)2dppz2+ or Ru(phen)2dppz2+ for 2 h causes
only a small change in the luminescence profile. When cells are
incubated with 10 µM Ru(DIP)2dppz2+, however, the luminescence
intensity of the cell population increases dramatically.
diameter
(Å)d
bpy
1.0
1.8
1.2
1.2
2.7
1.0
1.1
0.6
2.3
2.7
-2.50
-0.76
-0.43
-1.48
1.30
16.2
20.4
18.2
16.2
20.4
CO2Et-bpy
mcbpy
phen
DIP
a Excited at 488 nm; integrated emission at 600-620 nm; 10 µM Ru
was used, except for Ru(DIP)2dppz2+ in Tris buffer, where a lower
concentration was used due to poor solubility; emission values were scaled
accordingly. b Luminescence values with DNA were obtained at saturation.
c Cl- salt. d Diameters were estimated using Titan.
Uptake for the different Ru complexes may be compared based
upon the mean luminescence intensity of the cell population (Table
2). Below 1 µM, Ru(DIP)2dppz2+ is taken up appreciably above
background. At higher concentrations, Ru(bpy)2dppz2+, Ru(CO2-
Et-bpy)2dppz2+, and Ru(phen)2dppz2+ are taken up to some extent,
but even at 20 µM Ru, little luminescence is evident for Ru-
(mcbpy)2dppz.13 Washing with buffer reduces luminescence by 20-
50%, suggesting that, while some Ru is nonspecifically 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
Figure 2. Flow cytometry analysis of HeLa cells incubated with 10 µM
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.
evident by confocal microscopy (Figure 3).14 For Ru(DIP)2dppz2+
,
intense luminescence in the interior is apparent within 2 h. For
Ru(phen)2dppz2+ and Ru(bpy)2dppz2+, microscopy experiments also
show uptake into the cellular interior but, consistent with the flow
cytometry data, on a slower time scale (g4 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;15 without
protection from water through macromolecular binding, Ru quench-
ing 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.16 Flow cytometry experiments on nuclear preparations
isolated after incubation of cells with Ru(DIP)2dppz2+ 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
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10.1021/ja0677564 CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Table 2. Mean Luminescence Intensity of HeLa Cells Incubated
that the Ru complexes are stable to the intracellular environment;
no loss of luminescence is evident, as would be expected based
upon changes in complex coordination.
with Ruthenium Complex by Flow Cytometrya
Ancillary Ligands of RuL2dppz
concn
Flow cytometry, traditionally used to examine organic fluoro-
phores, 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.
(
µ
M)
bpy
CO2Et-bpy
mcbpy
phen
DIP
0.5
1
n.d.
n.d.
38
38
48 (27)
n.d.
n.d.
45
45
51 (29)
n.d.
n.d.
20
21
26 (19)
n.d.
n.d.
52
58
60
99
5
597
974 (571)
n.d.
10b
20b
111 (50)
a 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. b Samples washed
after incubation are shown in parentheses.
Acknowledgment. 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.
Supporting Information Available: Flow cytometry for cell nuclei.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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albumin fraction 5, and diluted to 1 × 106 cells/mL. The ruthenium
complexes were added to the cell suspensions at concentrations of 0.5-
10 µM and incubated for 2 h at ambient temperature. Dead cells were
stained with 1 µM To-Pro-3 (Molecular Probes), which enters cells having
compromised membranes.
Figure 3. Confocal microscopy of HeLa cells with Ru(DIP)2dppz2+. (Top)
Microscopy after incubation with 5 µM 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 µM Ru.
(13) The low luminescence from Ru(mcbpy)2dppz may be in part due to poor
nucleic acid binding by the neutral complex.
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)2dppz.17
There have been few systematic studies on the cellular uptake
of transition metal complexes reported.18-20 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 <1.20 Importantly, these data also establish
(14) Confocal microscopy of cells incubated with a Ru dimer has been reported.
See: O¨ nfelt, B.; Gostring, L.; Lincoln, P.; Norde´n, B.; O¨ nfelt, A.
Mutagenesis 2002, 17, 317.
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(16) After incubation of HeLa cells for 2 h with 5 µM Ru(DIP)2dppz2+, line
plot quantitation shows an average of 11% luminescence in the nucleus
compared to cytoplasm; for 10 µM Ru after 12 h, as in Figure 3 (bottom),
the ratio is ∼30%. Some contribution to fluorescence in the nucleus could
arise from cellular autofluorescence.
(17) The lower luminescence of Ru(mcbpy)2dppz with nucleic acids contributes
to its relatively poor luminescence in cells but cannot fully account for it.
(18) Jonas, S. K.; Riley, P. A. Cell Biochem. Funct. 1991, 9, 245.
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Biochem. 2004, 98, 73.
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