Peptide-Coated Platinum Nanoparticles with Selective Toxicity against Liver Cancer Cells

Article abstract of DOI:10.1002/anie.201813149

Peptide-stabilized platinum nanoparticles (PtNPs) were developed that have significantly greater toxicity against hepatic cancer cells (HepG2) than against other cancer cells and non-cancerous liver cells. The peptide H-Lys-Pro-Gly-dLys-NH₂ was identified by a combinatorial screening and further optimized to enable the formation of water-soluble, monodisperse PtNPs with average diameters of 2.5 nm that are stable for years. In comparison to cisplatin, the peptide-coated PtNPs are not only more toxic against hepatic cancer cells but have a significantly higher tumor cell selectivity. Cell viability and uptake studies revealed that high cellular uptake and an oxidative environment are key for the selective cytotoxicity of the peptide-coated PtNPs.

Full text of DOI:10.1002/anie.201813149

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Accepted Article
Title: Peptide-coated Platinum Nanoparticles with Selective Toxicity
against Liver Cancer Cells
Authors: Helma Wennemers, Michal S Shoshan, Thomas Vonderach,
and Bodo Hattendorf
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201813149
Angew. Chem. 10.1002/ange.201813149
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COMMUNICATION
Peptide-coated Platinum Nanoparticles with Selective Toxicity
against Liver Cancer Cells
Michal S. Shoshan,[a] Thomas Vonderach,[b] Bodo Hattendorf,[b] and Helma Wennemers[a]*
Abstract: Peptide-stabilized platinum nanoparticles (PtNPs) were
developed that have significantly greater toxicity against hepatic
cancer cells (HepG2) compared to other cancer cells and
noncancerous liver cells. The peptide H-Lys-Pro-Gly-DLys-NH2 was
identified by a combinatorial screening and further optimized to allow
for the formation of water-soluble, monodisperse PtNPs with
average diameters of 2.5 nm that are stable for years. In comparison
to cisplatin, the peptide-coated PtNPs are not only more toxic
against hepatic cancer cells but have a significantly higher tumor cell
selectivity. Cell viability and uptake studies revealed that high
cellular uptake and an oxidative environment are key for the
selective cytotoxicity of the peptide-coated PtNPs.
oxidation of Pt(0) to Pt(II). Split-and-mix libraries have proven
useful for the identification of short-chain peptides that control
the formation of PdNPs and AgNPs and endow them with
stability against aggregation in water.[17] We therefore started our
studies by a combinatorial screening for peptides that enable
PtNP formation with a library of maximally 153 = 3375 different
tri- and tetrapeptides (Figure 1a).[18] Upon incubating the library
with K2PtCl4 and addition of NaBH4 several dark beads were
observed (Figure 1a). The brown/black color indicates PtNPs
and suggests that the peptides on these beads had complexed
Pt(II) and enabled PtNP formation upon reduction. Analysis of
the peptides on several of the colored beads revealed H-Lys-
Pro-Gly-DLys-NH2 (1) as a consensus sequence.[19,20]
Hepatocellular carcinoma (HCC) is the sixth most frequent
cancer and the second leading cause of death from cancer
worldwide.[1] Sorafenib is the most commonly used FDA-
approved systemic drug for treating advanced HCC, but suffers
from low efficacy and severe side effects.[2,3] Thus, there is a
need for specific chemotherapeutics against HCC. Cisplatin is
an effective chemotherapeutic against many cancer cells but is
also toxic to healthy organs.[4,5] Platinum nanoparticles (PtNPs)
are promising alternatives to cisplatin and other Pt-based
complexes.[6-10] However, previous studies with various cancer
cell-lines neither showed improved cytotoxicity nor tumor
selectivity of PtNPs over cisplatin.[6-9]
a)
O
O
H
1. K2PtCl4
2. NaBH4
N
H2N
spacer
N
H
R1
AA1
153 = 3375 different peptides
R2
AA2
b)
NH2
NH2
H
N
H
N
N
Within PtNPs, cytotoxic Pt(II) ions[5] are masked as inert
Pt(0) atoms and oxidation of Pt(0) to Pt(II) is required to unleash
cytotoxicity. PtNPs should therefore have a particularly high
toxicity in cellular environments with a high oxidation potential.
Liver cancer cells have a higher oxidative state than other cells
due to high concentrations of reactive oxygen species.[11,12]
PtNPs should therefore be ideally suited to target HCC. A recent
report on pH-responsive polymer particles that were
functionalized with a targeting ligand for HCC and contained
PtNPs supported this assumption.[10] Upon entry of the
multicomponent system into HCC cells, the polymer matrix
disassembled and the released PtNPs exhibited cytotoxicity. We
envisioned that suitably functionalized PtNPs should allow for
selective toxicity against HCC cells without the need for
immobilization in a polymeric matrix. Such PtNPs have to be
water soluble, small,[13] and bear a coat of molecules that a)
prevent aggregation of the PtNPs, b) enable cellular uptake, and
c) allow for oxidation of Pt(0) to Pt(II) inside HCC cells.
Herein, we present peptide-coated PtNPs that are toxic
against hepatic cancer cells but not, or only barely, against other
cancer and noncancerous cells. The peptide, which was
identified in a combinatorial screening, endows the PtNPs with
high stability and monodispersity and can be functionalized with
additional moieties, e.g., glucose to enhance the uptake into
cancer cells.
Peptides are valuable for the formation of biocompatible
noble metal nanoparticles.[14-17] We therefore envisioned that
peptidic ligands would also enable the preparation of stable
PtNPs in aqueous solution and, moreover, could be tailored to
allow for their uptake into liver cancer cells and subsequent
H2N
CONH2
O
O
O
20 nm
5 nm
1
Figure 1. a) Combinatorial library and assay for PtNP formation; bead size
~100 µm. b) TEM micrographs of PtNP-1.
We then resynthesized 1 and evaluated its ability to form
and stabilize PtNPs in solution. Testing of different parameters
(e.g., stoichiometry, concentration, reducing agent) showed that
addition of NaBH4 (1.2 equiv) to a solution of the peptide (1
equiv) and Pt(II) ions (1 equiv) in a mixture of deionized water
and CH3CN (3:1) is optimal for the formation of small PtNPs with
a narrow size distribution and an average diameter of 1.9±0.8
nm (Figure 1b). The peptide-coated PtNPs proved stable for
months in the reaction mixture but aggregated upon dialysis,
which was necessary prior to the biological tests to remove
residual reagents. We therefore performed structure–activity
relationship (SAR) studies with analogues of 1 to elucidate
which functional groups are important for the formation of the
PtNPs and further improve the structure of the peptide to obtain
even more robust PtNPs.[19] These SAR experiments revealed
the following key features of 1 for the formation of stable PtNPs:
a) the amino groups of the lysine (Lys) residues and at the N-
terminus are essential, b) the C-terminal CO2H group must be
capped, and c) the two Lys residues must be in the i, i+3
positions. The absolute configuration of the backbone α-carbon
atoms plays a minor role. Replacement of the proline (Pro) and
glycine (Gly) residues by alanine led to stable NPs with lower
monodispersity. These findings suggest that the amino groups
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10.1002/anie.201813149
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complex Pt(II) ions and stabilize the resulting PtNP by
coordination to Pt(0). The results also suggest that the Pro and
Gly residues can be modified, e.g., for functionalization with
additional moieties. We therefore elongated the all-L-configured
diastereoisomer of 1 with an additional unit of Lys-Pro-Gly to the
heptamer 2 H-Lys-[Pro-Gly-Lys]2-NH2.[21] Reassuringly, small
PtNPs formed also in the presence of 2 with a narrow size
distribution (2.5±0.7 nm, Figure 2a). These nanoparticles did not
aggregate upon dialysis and the purified PtNPs proved to be
stable for >1 year even upon repeated lyophilization and
resuspension as judged by inspections by TEM. Such a high
level of stability is remarkable and typically only achieved with
ligands (e.g., thiols) that form covalent bonds with the noble
metal NP.[22]
a)
b)
100
OH
OH
HO
MeO
O
O
NH2
NH2
NH2
50
0
HN
N
O
H
N
H
N
H
N
H
N
N
NH2
H2N
O
O
O
O
O
O
O
2a
-1
0
log [mg/L]
1
c)
100
50
0
100
50
100
50
0
HepG2
HT-29
AML-12
-1
0
2
a)
b)
H-Lys-[Pro-Gly-Lys]2-NH2 (2)
-1
0
log [mg/L]
1
-1
0
log [mg/L]
1
2
0
log [mg/L]
1
2
100
50
0
cisplatin
sorafenib
PtNP-2
PtNP-2a
Figure 3. a) Structure of peptide 2a. b) Viability of human cancer cells treated
with PtNPs-2a: HepG2 (▲), HT-29 (■), MCF-7 (■), HeLa (▲), PC3 (■), A431
(⦁), A549 (■), A2780 (▲). c) Cell viability of HepG2 (left), HT-29 (middle), and
AML-12 (right) cells against sorafenib, cisplatin, PtNP-2, and PtNP-2a. Values
are mean±SD of ≥3 repeats performed each in triplicate.
20 nm
5 nm
-1
0
1
log [mg/L]
Figure 2. a) TEM micrographs of PtNP-2. b) Viability of human cancer cells
treated with PtNP-2: HepG2 (▲), HT-29 (■), MCF-7 (■), HeLa (▲), PC3 (■),
A431 (⦁), A549 (■), A2780 (▲).
We then compared the cytotoxicity of PtNP-2 and PtNP-2a
with that of the drugs cisplatin and sorafenib (Figure 3c). Aside
from the liver cancer cells (HepG2) we used colon cancer cells
(HT-29) for comparison with a cell-line that was not significantly
affected by the PtNPs. These experiments revealed that the
glucose-functionalized PtNP-2a have a comparable toxicity
against HepG2 cells (maximal inhibition 97%; IC50 = 2.4±0.6
mg/L) as sorafenib (maximal inhibition 93%; IC50 = 2.6±0.2
mg/L) and are significantly more toxic than cisplatin (maximal
inhibition 66%; IC50 = 3.1±0.4 mg/L, Figure 3c, left). Sorafenib
and cisplatin are also toxic against HT-29 cells (maximal
inhibition: 84% and 82%; IC50 = 5.3±0.4 mg/L and 4.2±0.5 mg/L).
In contrast, both PtNPs affected this cell-line to a much lesser
extent (maximal inhibition <5% (PtNP-2), 31% (PtNP-2a); IC50
>100 mg/L; Figure 3c, middle). Thus, whereas cisplatin and
sorafenib have comparable toxicities against different types of
cancer cells,[2-5,27] the peptide-coated PtNPs are significantly
more toxic against liver cancer cells than other cancer cells.
We also evaluated the toxicity of PtNP-2a, cisplatin and
sorafenib towards noncancerous mouse liver cells (AML-12).[28]
Cisplatin (IC50 = 8.3±0.5 mg/L, maximal inhibition ≥ 64%) and
sorafenib (IC50 = 9.1±0.6 mg/L, maximal inhibition ≥ 86%) are
toxic to these noncancerous cells,[29] but the peptide-coated
PtNPs have only a small effect on their viability (IC50 >100 mg/L;
maximal inhibition 25%, Figure 3c, right). Thus, the PtNPs are
not only more toxic against hepatic compared to other cancer
cells, but also differentiate between noncancerous and
carcinogenic liver cells.
Next, we examined the effect of PtNP-2 on the viability of
hepatocellular carcinoma cells (HepG2) and other human
carcinoma cells that included colon (HT-29), breast (MCF-7),
cervical (HeLa), prostate (PC3), epidermoid (A431), lung (A549),
and ovarian (A2780) cells. Different amounts of the PtNPs were
incubated with the cells for three days and their viability was
then determined by MTT assays.[23] For the liver cancer cells
(HepG2) an IC50 value of 2.9±0.3 mg/L and a maximal inhibition
value of 79% were observed (Figure 2b, orange). None of the
other carcinoma cells were affected to a comparable extent by
the PtNP-2 under the same conditions. This result demonstrates
the selective toxicity of the peptide-coated PtNPs against liver
carcinoma cells.
Cancer cells have a higher glucose metabolism and more
glucose transporters on their surface than noncancerous cells.[24]
We envisioned that functionalization of the peptide with glucose
could enhance the cellular uptake and effective cytotoxicity of
the peptide-coated PtNPs. We therefore prepared peptide 2a
that bears a glucose moiety and used it to form PtNPs
(Figure 3a).[25] The PtNPs have similar stability (>1 year), size
and monodispersity (2.7±0.8 nm), and coverage of the peptide
on the NP surface (~4.8 peptides/nm2) as those formed in the
presence of peptide 2.[19,22] This result shows the tolerance of
the PtNP formation process toward a functional moiety on
peptide 2. Cell viability tests with PtNP-2a showed that they
inhibit the growth of hepatic cancer cells HepG2 almost
completely (maximal inhibition 97%; IC50 = 2.4±0.6 mg/L;
Figure 3b). In contrast, the other cancer cell-lines were not
affected to nearly the same extent, which further highlights the
specific toxicity of the PtNPs against liver cancer cells.[26]
The observed toxicity profile could be due to higher uptake
of the PtNPs, particularly those functionalized with glucose, or
increased oxidation of Pt(0) to Pt(II) inside the hepatic cancer
cells compared to other cells; or a combination of both effects.
Accordingly, we quantified the cellular uptake of PtNP-2, PtNP-
2a, and cisplatin into HepG2 and HT-29 cells with inductively
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10.1002/anie.201813149
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COMMUNICATION
coupled plasma mass spectrometry (ICP-MS, Table 1).[30] These
studies revealed higher uptake of both types of PtNPs (up to 20-
fold) than of cisplatin into HepG2 and HT-29 cells (Table 1,
entries 1 and 2). PtNP-2a have an even higher cellular uptake
(2–3-fold) than PtNP-2 as expected based on the increased
glucose metabolism in cancer cells.[24] The same amount of
cisplatin (5.5 ng Pt/mg cells) was internalized in HepG2 and HT-
29 cells, which corroborates the lack of cell-type selectivity of
cisplatin. In contrast, the uptake of the peptide-coated PtNPs
PtNPs. The peptide-coated PtNPs are highly toxic against
hepatic cancer cells but do not affect the viability of other cancer
or noncancerous cells to nearly the same extent. To the best of
our knowledge, these PtNPs have the highest cytotoxicity
combined with selectivity for hepatic cancer cells achieved so far.
Cellular uptake combined with cell viability studies revealed that
the combination of high cellular uptake and an oxidative
environment is key for the cytotoxicity of PtNPs. These results
open exciting prospects for the development of PtNP-based
into HepG2 cells is twice as high as their uptake into HT-29 cells, therapeutics.
presumably due to the higher metabolism in liver compared to
colon cells.[31] The highest amount of Pt (107±14 ng Pt/mg cells)
was detected in the HepG2 cells after incubation with the
Acknowledgements
glucose-functionalized PtNP-2a (Table 1, entry 1).
Table 1. Quantification of platinum inside cells
[Pt] ng/ cells mg[a]
This project has received funding from the European Union’s
Horizon 2020 research and innovation programme under the
Marie Sklodowska-Curie grant agreement No 705970. We also
thank the Swiss National Science Foundation for support by
Sinergia Grant CRSII2_160805. We are grateful to Leyla
Hernandez for assistance in cell culture. We thank the Scientific
Center for Optical and Electron Microscopy (ScopeM) and Prof.
Detlef Günther (ETH Zurich) for access to high resolution TEM
and ICP-MS instruments, respectively.
Entry
Cell-line
Cisplatin
PtNP-2
PtNP-2a
Whole cells[b]
1
HepG2
5.3 ± 0.2
5.5 ± 1.4
40 ± 1
19 ± 5
107 ± 14
61 ± 7
2
HT-29
Nuclei[c]
Keywords: peptides • Platinum nanoparticles • Cytotoxicity •
Selectivity • Hepatocellular carcinoma
3
4
HepG2
HT-29
n.d.[d]
n.d.[d]
29 ± 5 (72%)
5 ± 3 (26%)
71 ± 10 (66%)
16 ± 7 (27%)
[1]
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The higher cellular uptake of the PtNPs correlates well with
their higher toxicity compared to that of cisplatin in the HepG2
cells (Figure 3c, left). This is not the case in HT-29 cells. Despite
their higher internalization, the PtNPs are significantly less toxic
against HT-29 cells than cisplatin (Figure 3c, middle). These
results show that the amount of Pt alone does not determine the
cytotoxicity of PtNPs but that the environment inside the cells
plays a key role. In HT-29 cells, the PtNPs with neutral Pt(0)
atoms are barely toxic, consistent with toxic Pt(II) ions being
formed more readily in the oxidative environment of the hepatic
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(Table 1, entries 3 and 4).[19] In the HepG2 cells, ~70 % of the Pt
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enabled the development of the peptide H-Lys-[Pro-Gly-Lys]2-
NH2 as an additive for the formation of small, monodisperse
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10.1002/anie.201813149
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previously developed peptides for PtNP formation (ref. 15).
[21] Further elongation to the decamer H-Lys-[Pro-Gly-Lys]3-NH2 (3) led to
PtNPs with comparable size, monodispersity, and stability as PtNP-2.
PtNPs formed in the presence of tredecamer H-Lys-[Pro-Gly-Lys]4-NH2
(4) were polydisperse and bigger. See the supporting information for
details.
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proportion of atoms at the surface becomes larger the smaller the NPs
are.
[22] EDX and STEM analysis confirmed the formation of ~2.5 nm sized
PtNP-2. TGA, ICP-MS and ζ potential analysis revealed a coverage of
~4.5 peptides per nm2 on the PtNPs and a ζ potential of +3.6 ± 0.8 mV.
See the supporting information for details.
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presumably since skin cancers have also
concentration of ROS.
a comparatively high
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4231.
[29] For previous studies on the toxicity of sorafenib against AML-12 cells,
see: Y. L. Chen, J. Lv, X. L. Ye, M. Y. Sun, Q. Xu, C. H. Liu, L. H. Min,
H. P. Li, P. Liu, X. Ding, X. Hepatology 2011, 53, 1708.
[17] a) S. Corra, U. Lewandowska, E. M. Benetti, H. Wennemers, Angew.
Chem. Int. Ed. 2016, 55, 8542. b) K. Belser, T. V. Slenters, C.
Pfumbidzai, G. Upert, L. Mirolo, K. M. Fromm, H. Wennemers, Angew.
Chem. Int. Ed. 2009, 48, 3661.
[30] Quantification of the Pt-amount in the stock solutions of the PtNPs and
cisplatin by ICP-MS ensured that the same amount of Pt was added to
the cells, see the supporting information for details.
[18] Amino acids included in the library: AAn: Pro, DPro, Arg, Lys, His, Trp,
Asp, Glu, Asn, Gln, Ser, Thr, Tyr, Phe, Val, Ala. spacer: Pro, ProAib,
ProGly, Gly, (1R,2R)Achc, (1S,2S)Achc, Leu, DLeu, Phe, DPhe, Val,
DVal, Ala, Ahx. See ref. 17 for details on the library and for the method
of encoded split-and-mix libraries: M. H. J. Ohlmeyer, R. N. Swanson, L.
W. Dillard, J. C. Reader, G. Asouline, R. Kobayashi, M. Wigler, W. C.
Still, Proc. Natl. Acad. Sci. U S A 1993, 90, 10922.
[31] a) H. G. Hers, Annu. Rev. Biochem. 1976, 45, 167. b) G. Musso, R.
Gambino, M. Cassader, Prog. Lipid Res. 2009, 48, 1.
[32] The necessity of an oxidative environment for unleashing the toxicity of
PtNPs was further supported by incubating PtNP-2 with HT-29 cells in
the absence and presence of H2O2. Whereas PtNPs-2 are barely toxic,
toxicity was observed upon addition of H2O2 (at a concentration which
is non-toxic) against HT-29 cells, see the supporting information for
details.
[19] For details, see the Supporting Information.
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10.1002/anie.201813149
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Michal S. Shoshan, Thomas Vonderach,
Bodo Hattendorf, Helma Wennemers*
Page No. – Page No.
Peptide-coated Platinum
Nanoparticles with Selective Toxicity
against Liver Cancer Cells
Selective Toxicity. Peptide-stabilized, monodisperse platinum nanoparticles were
developed that selectively kill liver cancer cells over other cancer and non-
cancerous cells. The peptide was identified in a combinatorial screening and
enables high stability of the PtNPs, their cellular uptake, and release of toxic Pt(II)
ions in an oxidative environment.
This article is protected by copyright. All rights reserved.