Cellular uptake quantification of metalated peptide and peptide nucleic acid bioconjugates by atomic absorption spectroscopy

Article abstract of DOI:10.1002/anie.200703994

(Figure Presented) The metal does the trick! Atomic absorption spectroscopy (AAS) of cobalt atoms is used as an accurate method to determine the cellular uptake and nuclear localization of metal bioconjugates. Surprisingly, the PNA conjugates show highest

Full text of DOI:10.1002/anie.200703994

Angewandte
Chemie
DOI: 10.1002/anie.200703994
Cellular Uptake
Cellular Uptake Quantification of Metalated Peptide and Peptide
Nucleic Acid Bioconjugates by Atomic Absorption Spectroscopy**
Srecko I. Kirin, Ingo Ott, Ronald Gust, Walter Mier, Thomas Weyhermüller,
and Nils Metzler-Nolte*
Advances in molecular biology have allowed the design of
targeted biopolymers such as proteins and antibodies to
become a reality in drug discovery. Research into cell uptake
and tissue selectivity of such macromolecules is one of the
most important areas in drug development, for instance in the
design of tumor-specific antiproliferative agents. Interest-
ingly, the principles observed for cell uptake also apply to the
intracellular distribution of exogenous compounds.[1] For
example, a compound that is designed to interfere with
DNA or DNA-processing enzymes should ideally be located
exclusively in the nucleus of a cell. When properly localized,
undesired side effects that might result from interaction with
other targets in the cell are minimized. Sophisticated techni-
ques are therefore warranted to allow insight into the
intracellular localization of drugs. The uptake and transport
of metal ions in nature is tightly controlled and regulated by
so-called metallochaperones.[2] The trafficking of copper ions,
which is associated with a number of malfunctions such as
Menkeꢀs and Wilsonꢀs disease, is particularly well-investi-
gated.[3] With the exception of antitumor-active platinum
complexes, the intracellular localization of exogenous metal
complexes has hardly been explored. Indeed, the design of
artificial metallochaperones for the directed intracellular
delivery of metal complexes has only recently been
addressed.[4,5] Since DNA, which codes for the genetic
information of the cell, is located in the cellular nucleus, it
would be a particularly sensitive target for toxic metal ions.
The nucleus of eukaryotic cells is protected by the nuclear
membrane. However, the pores in this membrane are large
enough to allow the passive diffusion of small molecules.
Alberto and co-workers were able to demonstrate the
accumulation of 99mTc ions, which may cause DNA cleavage
by short-range Auger electrons, near the cellular DNA in a
conjugate with the intercalator pyrene.[6] A more rational
approach is to use the cellular machinery for the accumu-
lation of metal ions in the nucleus. Nuclear localization signals
(NLS) are small peptides which, upon binding to cargo
proteins, translocate into the cellular nucleus.[7] The simian
virus SV40 antigen NLS has been extensively studied. It has
the primary sequence Pro-Lys-Lys-Lys-Arg-Lys-Val and has
been used in nuclear translocation studies of a variety of
cargos, e. g. proteins, DNA, RNA, peptide nucleic acid
(PNA), and even gold nanoparticles.[8]
We recently reported a study on the nuclear localization
of cobaltocenium NLS bioconjugates using fluorescence
microscopy.[9] It might be argued that the fluorescent dye,
which is necessary for detection, might influence the cellular
uptake and nuclear localization of the conjugate. A more
elegant approach would be direct detection of the metal
complex. To this end, atomic absorption spectroscopy
(AAS),[10,11] inductively coupled plasma mass spectrometry
(ICP-MS),[12,13] X-ray fluorescence,[14,15] and visible-light fluo-
rescence microscopy have been suggested for nonradioactive
metal ions and their complexes.[16,17] The latter method is not
generally applicable as it requires that the metal complex
itself is fluorescent. X-ray fluorescence, on the other hand,
requires a powerful synchroton source and sophisticated
equipment to achieve the required spatial resolution.[15]
Moreover, the cells need to be fixed, which may cause
intracellular redistribution of compounds, as has recently
been demonstrated for small, cationic cell-penetrating pep-
tides.[18] This leaves AAS and ICP-MS as the most feasible
and general methods for direct studies of metal-ion distribu-
tion inside living cells.
[*] Dr. S. I. Kirin, Prof. Dr. N. Metzler-Nolte
Department of Chemistry and Biochemistry
University of Bochum
Universitätsstrasse 150, 44801 Bochum (Germany)
Fax: (+49)234-321-4378
E-mail: nils.metzler-nolte@ruhr-uni-bochum.de
Homepage: http://www.rub.de/ac1
Dr. I. Ott, Prof. Dr. R. Gust
Institute of Pharmacy
Free University Berlin
Königin-Luise-Strasse 2 + 4, 14195 Berlin (Germany)
Dr. W. Mier
Department of Nuclear Medicine
University HospitalHeidebl erg
Im Neuenheimer Feld 400, 69120 Heidelberg (Germany)
Dr. T. Weyhermüller
Max Planck Institute for Bioinorganic Chemistry
Stiftstrasse 34–36, 45470 Mülheim an der Ruhr (Germany)
[**] This work is supported by the German Research Foundation (DFG)
through funding of a Research Unit “Biological Function of
Organometallic Compounds” (FOR 630). Synthesis and partial
chemicalcharacterization of the conjugates was carried out at the
Institute of Pharmacy and Molecular Biotechnology (IPMB) at the
University of Heidelberg. The authors are grateful to Dr. Ulrich
Schatzschneider for measuring the ESI-MS spectra.
Herein, we describe the use of peptides and PNAs as
biological vectors to enhance cellular uptake and nuclear
localization of cobalt complexes. AAS is the method of choice
to directly quantify uptake and cellular localization of these
conjugates. Cobalt was used in this study because it is not
particularly toxic,[19] its natural background is low, and an
excellent comparison is available in the form of cyanocobal-
amine (vitamin B12). The bis(picolyl)amine (bpa) ligand 1 was
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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chosen because it forms stable complexes with a variety of
the Supporting Information, Figure S1). The difference in
melting temperature DTm = 6.38C can be ascribed to an
attractive electrostatic interaction between the positively
charged NLS peptide and the negatively charged overhanging
DNA at the 5’-end in duplex 6·7.
metal ions, the properties of which have been extensively
studied.[20,21] Moreover, carboxylato-functionalized bpa
ligands 2 (ester) and 3 (free acid) are readily available
(Scheme 1).[5,22]
The bpa ligand readily forms complexes with divalent
metal ions. The [Co(bpa)]complexes studied herein were
obtained in situ by mixing equimolar amounts of bpa ligand
conjugates and Co(NO3)2. To gain insight into the coordina-
tion chemistry and structural parameters of these complexes,
the CoII complex 2Co of the bpa ester (see also Scheme 1) was
isolated and characterized by X-ray crystallography. An ball-
and-stick representation of 2Co is shown in Figure 1. In the
Scheme 1. Synthesis of the compounds used in this study, see Table 1
for details of the bioconjugates 4–6. SPPS=solid-phase peptide syn-
thesis, Ahx=w-aminohexanoic acid, NLS=Pro-Lys-Lys-Lys-Arg-Lys-Val-
NH2, PNA=tgttatcc. All metal complexes are denoted with the sub-
script “Co”.
Figure 1. Crystalstructure of 2Co. Hydrogen atoms are omitted for
clarity. Selected bond lengths [Š] and angles [8]: Co(1)–N(1) 2.074(6),
Co(1)–N(8) 2.197(5), Co(1)–N(15) 2.109(5), Co(1)-O(31) 2.140(6),
Co(1)–O(41) 2.120(5), Co(1)–O(42) 2.208(5), N(1)-Co(1)-N(15)
111.7(2), N(8)-Co(1)-N(1) 77.6(2), N(8)-Co(1)-N(15) 76.54(19), N(8)-
Co(1)-O(41) 85.87(18), N(8)-Co(1)-O(31) 157.7(2), N(8)-Co(1)-O(42)
108.20(18), O(31)-Co(1)-O(42) 89.69(19), O(41)-Co(1)-O(42)
59.22(19).
The synthesis of the conjugates studied is shown in
Scheme 1. The oligomers bpa-NLS 4, bpa-PNA 5, and bpa-
PNA-NLS 6 were prepared by standard 9-fluorenylmethoxy-
carbonyl (Fmoc) solid-phase peptide synthesis (SPPS) using
the bpa acid 3. All new conjugates were purified by reversed-
phase HPLC and characterized by MALDI-TOF mass
spectrometry.[5,9,23] The sequences of the oligomers are given
in Table 1.
solid state, 2Co is a discrete mononuclear complex with a
hexacoordinated Co center. The N3O3 coordination is ach-
ieved by three nitrogen atoms of the bpa ligand and three
oxygen atoms of two nitrate counterions. The bpa ligand
coordinates in a facial fashion, and the angle between the two
planes of the pyridine rings is 72.48.
Table 1: Composition of the compounds used in this study.[a]
No.
Abbreviation
bpa
Sequence/Compound
Cellular uptake experiments on the cobalt complexes
were performed in HT-29 colon carcinoma cells after
exposure to 50 mm of the compounds over an incubation
period of 6 or 24 h (Figure 2a). The uptake of metal
compounds into cells and nuclei was then quantified by
determination of the cobalt content of isolated cells or
nuclear pellets by AAS. This value was normalized to the total
protein content of the cells (pmol cobalt per mg cellular
protein, Figure 2a). In the cell uptake studies, cyanocobala-
min (8, vitamin B12) was used as a reference. In separate
experiments, the nuclei were separated by density-gradient
centrifugation, and the cobalt content of the nuclei was
determined by AAS after 24 h. In this case, the amount of Co
was normalized to the nuclear protein content (pmol cobalt
per mg nuclear protein, Figure 2b). The ratio of nuclear to
cellular protein content may vary from preparation to
preparation. While the cobalt-to-protein ratio is reliable and
reproducible for each determination, there is no universal
1
2
3
4
Bis(picolyl)amine
bpa-CH2-(p-C6H4)-C(O)-OMe
bpa-CH2-(p-C6H4)-C(O)-OH
bpa-CH2-(p-C6H4)-C(O)-Ahx-Pro-Lys-Lys-Lys-
Arg-Lys-Val-NH2
bpa-NLS
5
6
bpa-PNA
bpa-PNA-NLS
bpa-CH2-(p-C6H4)-C(O)-Ahx-tgttatcc-Lys-NH2
bpa-CH2-(p-C6H4)-C(O)-Ahx-tgttatcc-Gly-
Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-NH2
5’-TAGGGATAACAGGGTAAT-3’
7
DNA18
[a] Small letters (a, c, t, g) indicate PNA and capital letters DNA (A, C, G,
T) monomer units; the three-letter code is used for amino acids. All
peptide sequences are written from N to C terminus (left to right). The
complemantary (to PNA 5 and 6) part of DNA 7 is shown in bold.
The PNA-containing oligomers 5 and 6 were also
characterized by UV melting experiments with a comple-
mentary DNA oligomer 7.[24] The results show a hybridization
in both 5·7 (Tm = 29.78C) and 6·7 (Tm = 36.18C) duplexes (see
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series 2Co < 4Co < 5Co ꢀ 6Co. Absolute differences are even
more pronounced for nuclear uptake. Thus, 5Co was found in
approximately 14-fold higher concentration in the nuclei than
2Co, but only about four times more in the whole-cell analysis.
On the basis of distinct cellular parameters such as the
mean cellular protein content and mean cellular volume, the
molar concentrations can be estimated from the metal
concentration (pmol metal per mg protein).[25] For the com-
pounds with the highest uptake rates (5Co and 6Co after 24 h),
intracellular concentrations greater than 70 mm were calcu-
lated, whereas only 6 mm are reached for the complex with the
lowest cellular accumulation (2Co after 6 h). All other com-
pounds also reached cellular levels well below 50 mm, which is
the extracellular concentration during the incubation period.
Intracellular concentrations greater than 50 mm may indicate
an active cellular uptake process for conjugates 5Co and 6Co.
This result is of special interest, as the intracellular levels of
even very active drugs, such as cisplatin, hardly exceed the
extracellular concentration.[13,26] As we reported previously,
the cellular uptake of metal-based drugs can be significantly
influenced by the properties of the ligand and the charge of
the metal atom.[27] In this context, the somewhat low overall
uptake of the target compounds presented herein may be the
consequence of the hydrophilic nature of the peptide-
containing ligands and the positive charge of the cobalt
complexes after replacement of the nitrato ligands by water
upon dissolution. Interestingly, the uptake was significantly
improved by the presence of the PNA sequence in the ligands.
This aspect is surprising in light of earlier reports on rather
poor uptake of PNA oligomers into mammalian cells and will
be further discussed below.
It is conceivable that increased uptake results from
cytotoxic effects of the conjugate. Therefore, cytotoxicity
experiments were performed on 2Co and 4Co. No toxic effects
were noted up to the highest concentration of 100 mm (data
not shown). This finding indicates that metal uptake into the
cells and nuclei was not facilitated by apoptotic or necrotic
processes, which might have caused higher permeability of the
nuclear membranes.
Several conclusions can be drawn from the above data.
First of all, AAS as a detection method is selective for the
metal and has an appropriate sensitivity. Typical experimental
values are on the order of 0.2–0.6 mm Co, and thus at least ten
times higher than the detection limit of the method.
Significantly different metal concentrations are observed
across the bioconjugates. Therefore, we conclude that the
peptides remain coordinated to the Co ions throughout the
study. Significantly improved uptake is observed for the NLS
conjugates over the simple [Co(bpa)]complex. This result
may seem somewhat surprising, as the NLS peptide alone is
not a good vector for cellular uptake in bioconjugates.[28] On
the other hand, we have shown in previous work that
attachment of cationic organometallic complexes to the
N terminus of NLS drastically improves the uptake efficiency
of the conjugates.[9] Moreover, we show that the nuclear
localization properties of the NLS peptide are not affected by
the attached metal complex.
Figure 2. a) Cellular uptake into HT-29 cells after 6 and 24 h exposure
to 50 mm solutions of 2Co, 4Co, 5Co, 6Co, and 8Co (n=2); b) uptake into
the nuclei of HT-29 cells after 24 h exposure to 50 mm solutions of 2Co,
4Co, 5Co, and 6Co (n=2). Please note the different y axis, which refers to
cellular protein mass in (a) but to nuclear protein content in (b). See
the text and ExperimentalSection for detaisl.
conversion factor between the cobalt content of Figure 2a
and Figure 2b. As a rough estimate, the nuclear protein
content is on the order of 10–20% of the total cellular protein
mass.
The detection limits of cobalt in our AAS experiments
were 0.014 mm (0.82 mgLÀ1) for a typical cell suspension as
used for cellular uptake studies and 0.023 mm (1.35 mgLÀ1) for
suspensions of nuclei. The results in Figure 2 are presented as
mean values calculated from the data of two independent
experiments (different day, different passage of cells, freshly
prepared solution of compounds). The higher detection limit
for Co in the nuclear fraction is due to the higher systematic
background in all measurements. This background results
from the fairly high phosphate content of the DNA, which is,
naturally, only present in nuclear preparations.
For the purpose of this study, vitamin B12 (8) may be
regarded as a stable, nontoxic Co complex and is thus a more
appropriate comparison for cellular uptake studies than, for
example, simple metal salts would be. Indeed, Co(NO3)2 was
internalized by HT-29 cells in much higher amounts than all
compounds used herein, presumably by binding to transferrin
or by uptake through ion channels (data not shown). Neither
mechanism is available to the metal complexes, and therefore,
simple metal salts do not serve as relevant comparisons.
Figure 2 shows that the amount of Co inside the cells is almost
doubled between 6 and 24 h, independent of the nature of the
Co compound. Interestingly, all bpa-containing compounds
were found in higher amounts in the cells than vitamin B12.
Cellular uptake of the [Co(bpa)]complexes increases in the
It may further seem surprising that PNA conjugates are
internalized even better than the [Co(bpa)-NLS]conjugate.
Angew. Chem. Int. Ed. 2008, 47, 955 –959
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The combination of NLS peptide and PNA oligomer, on the
ex vivo into the nucleus of living cells, where they could exert
their activity on the cellular DNA in vivo.
other hand, does not yield any improvement in uptake
efficiency over PNA alone. The properties of the conjugate
are thus dominated by the PNA part of the molecule, not the
NLS peptide, at least with respect to cellular uptake. Limited Experimental Section
Full experimental details are provided in the Supporting Information.
cellular uptake has seriously hampered the use of PNA
oligomers as antisense agents, and numerous solutions to this
problem were proposed.[28,29] Krämer and co-workers
reported enhanced cellular uptake of PNA oligomers with
N-terminally attached Zn2+ terpyridine complexes.[17] These
results were, however, obtained by flow cytometry and
fluorescence microscopy of organic dyes and therefore
constitute indirect, nonquantitative evidence. In a compre-
hensive study with numerous peptides, Nielsen and co-
workers recently evaluated the uptake efficiency of PNA–
peptide conjugates by a splice-correction assay.[28] In terms of
antisense PNA delivery into cells, this work is certainly most
relevant, because it uses a biological effect as the read-out. On
the other hand, antisense efficiency in this system will depend
on additional factors, such as RNA binding and the response
of the cellular machinery. Thus, even this system is again only
an indirect one to determine the numbers of molecules that
were internalized into the cell.
There is a significant difference between the various
conjugates studied herein with respect to nuclear localization.
Our data show that enhanced transport of metals into nuclei is
possible by choice of appropriate peptide vectors. While the
ratios 4Co/6Co and 5Co/6Co are similar in the nuclei and whole
cells, the situation is very different for the nonpeptide
complex [Co(bpa)], for which the ratio 2Co/6Co is 23% in the
whole cells but only 7% in the nuclei. Clearly, bioconjugation
with either PNA or NLS (or both) favors nuclear localization.
This result is in good agreement with recent studies reporting
the nuclear accumulation of NLS conjugates with cobaltoce-
nium, nucleotide, and platinum moieties.[9,30] Again, there is
no difference between the PNA conjugates 5Co and 6Co, but
both show a higher nuclear localization that the NLS
conjugate 4Co. Finally, only a relatively small fraction (less
than 10%) of the conjugates reaches the nucleus. This result,
however, is in line with findings for other metal complexes
like cisplatin, of which also only about 5% is found in the
nucleus of treated cells.
4: C66H107N19O9, Mr = 1310.68 gmolÀ1, MS (ESI-pos.): m/z = 655.7
[M+2H]2+, 437.26 [M+3H]3+; 5: C118H151N47O30, M = 2707.76 gmolÀ1
,
MS (MALDI): m/z = 2707.1 [M+H]+; 6: C156H221N61O38, M =
3558.81 gmolÀ1, MS (MALDI) = 3558.2 [M+H]+.
X-ray crystallography: Crystal data for 2Co: Pink crystals of 2Co
,
C21H21CoN5O8, Mr = 530.36 gmolÀ1 0.12 ” 0.12 ” 0.06 mm3, mono-
,
clinic, space group P21/c, Z = 4, a = 9.8665(6), b = 14.2680(7), c =
16.7896(8) Š, b = 106.13(1)8, V= 2270.5(2) Š3, MoKa radiation (l =
0.71073 Š), m(MoKa) = 6.45 mmÀ1
,
1 = 1.551 gcmÀ3
,
T= 100(2) K,
11041 reflections measured, 3386 independent reflections (Rint
=
0.043), 3212 observed reflections (Fo > 4s(Fo)), 2qmax = 62.858, R =
0.0897 (Fo > 4s(Fo)), R = 0.0946 (all data), wR = 0.1661 (Fo >
4s(Fo)), 311 parameters, refinement against F2, ShelXTL 6.14
Bruker AXS program suite. CCDC-648421 contains the supplemen-
tary crystallographic data for this paper. These data can be obtained
free of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
Uptake into HT-29 cells and nuclei: HT-29 human colon
carcinoma cells were maintained in Eagleꢀs MEM cell culture
medium (Sigma, Germany) supplemented with NaHCO3 (2.2 gLÀ1),
sodium pyruvate (110 mgLÀ1), gentamycin (50 mgLÀ1), and 10 vol%
fetal calf serum according to standard procedures. For uptake studies
the cells were grown in 75-cm2 culture flasks at 378C in an atmosphere
of 5% CO2 and 95% air until at least 70% confluency. The medium
was replaced with 10 mL medium containing the freshly prepared
complexes in a concentration of 50 mm. Solutions of the metal
complexes were prepared by mixing equimolar amounts of 50 mm
solutions of compounds 4–6 and Co(NO3)2 in water. 10 mL of this
mixture was added to 10 mL MEM to give the desired concentration
with MEM. After further incubation for 6 or 24 h hours the cells were
detached by trypsinization, resuspended in 10 mL phosphate-buf-
fered saline pH 7.4 (PBS), pelleted by centrifugation (2000 rpm,
5 min), and washed twice with PBS. For cell uptake studies the
isolated pellets were resuspended in 1.0 mL twice-distilled water and
lysed using a sonotrode (Bandelin Sonoplus GM 70). The cobalt
content and protein content of the lysates was determined according
to a reported procedure.[10] The molar cellular concentrations were
calculated according to the literature.[25] For quantification of the
uptake into the nuclei, the isolated pellets were treated as previously
described.[11]
Cytotoxicity experiments: Cell growth inhibitory effects were
evaluated as previously described.[25]
In summary, this work investigates the use of peptides and
peptide nucleic acid oligomers (PNA) as vectors for the
cellular uptake and nuclear delivery of metal complexes. AAS
is used as the metal-specific detection method. Compared to
previous studies, which use indirect methods such as fluores-
cence spectroscopy or splice-correction assays, metal-specific
AAS is a method which allows accurate determination of
intracellular concentrations of the conjugates. It is thus most
suited to establish uptake efficiencies for any metal-contain-
ing bioconjugate and is applied herein to study the cell uptake
of PNA oligomers and peptides for the first time. For the best
systems in this study, an accumulation 150% higher than in
the cell culture medium was achieved.
Received: August 30, 2007
Revised: October 9, 2007
Published online: December 18, 2007
Keywords: cobalt · medicinal chemistry · N ligands ·
.
nucleic acids · peptides
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