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N-Heterocyclic Carbene-Polyethyleneimine Platinum Complexes with Potent in vitro and in vivo Antitumor Efficacy. Neila Chekkat, Georges Dahm, Edith Chardon, May Wantz, Justine Sitz, Marion Decossas, Olivier Lambert, Riccardo Rubbiani, Benoit Frisch, Gilles Gasser, Gilles Guichard, Sylvie Fournel, and Stephane Bellemin-Laponnaz Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00320 • Publication Date (Web): 26 Jul 2016 Downloaded from http://pubs.acs.org on August 5, 2016
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Bioconjugate Chemistry
N-Heterocyclic Carbene-Polyethyleneimine Platinum Complexes with Potent in vitro and in vivo Antitumor Efficacy
Neila Chekkat,‡ Georges Dahm,§ Edith Chardon,§,† May Wantz,‡ Justine Sitz,‡ Marion Decossas,† Olivier Lambert,† Benoit Frisch,‡ Riccardo Rubbiani,¶ Gilles Gasser,¶ Gilles Guichard,† Sylvie Fournel,*,‡ and Stéphane Bellemin-Laponnaz*,§
‡
Faculté de Pharmacie, Université de Strasbourg-CNRS UMR 7199, 74 Route du Rhin, BP
60024, 67401 Illkirch cedex (France) E-mail:
[email protected] §
Institut de Physique et Chimie des Matériaux de Strasbourg Université de Strasbourg-CNRS
UMR 7504, 23 rue du Loess, BP 43, 67034 Strasbourg cedex 2 (France) E-mail:
[email protected] †
Université de Bordeaux, CBMN, UMR 5248, Institut Européen de Chimie et Biologie, 2 rue
Robert Escarpit, 33607 Pessac (France) and CNRS, CBMN, UMR 5248, 33600 Pessac (France) ¶
Department of Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich
(Switzerland)
N.C. and G.D. contributed equally to this work.
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ABSTRACT
The current interest for platinum N-heterocyclic carbene complexes in cancer research stems from their impressive toxicity reported against a range of different human cancer cells. To date, the demonstration of their in vivo efficacy relative to established platinum-based drugs has not been specifically addressed. Here, we introduce an innovative approach to increase NHC-Pt complex potency whereby multiple NHC-Pt(II) complexes are coordinated along a polyethylenimine polymer (PEI) chain. We show that such NHC-Pt(II)-PEI conjugates induce human cancer cell death in vitro and in vivo in a xenograft mouse model with no observable side effects in contrast to oxaliplatin. Additional studies indicate nucleus and mitochondria targeting and suggest various mechanisms of action compared to classical platinum-based anticancer drugs.
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Introduction
Cis-diaminedichloroplatinum (II), best known as cisplatin and its Pt(II) analogues represent the most widely used chemotherapeutic drugs worldwide for the treatment of different types of cancers (i.e. testicular, ovarian, head neck, colorectal, lung cancer and non-Hodgkin lymphoma).1 Over 50% of tumors are currently treated with platinum-based drugs alone or co-administered with other chemotherapeutic agents or in combination with surgery and/or radiotherapy.2-4 In spite of their efficiency, the clinical applications of Pt-based drugs are limited by severe side effects, which include nephrotoxicity, neurotoxicity, and ototoxicity. Furthermore, platinum treatment is frequently associated with intrinsic or acquired cell resistances. These limitations are mainly due to poor selectivity and stability of the drugs itself.5 To circumvent the drawbacks of cisplatin and its derivatives, various strategies have been developed. These strategies are based either on the development of new platinum complexes that display different DNA-binding modes in the nucleus or have alternative targets, including mitochondria and proteins or on the improvement of drug delivery to permit accumulation of the platinum drug in tumor sites.6-15 In recent years, a number of research groups highlighted the potential of N-Heterocyclic Carbenes (NHC) as new chemical structures for the development of metal-based drugs.16-29 These highly σ-donating ligands form stable transition metal complexes, which might be a key requirement for biological applications.30 Indeed, the lack of in vivo efficacy and side effects of some metal-based anticancer drugs are strongly correlated with their instability under physiological conditions.31 The cytotoxic activity of a variety of NHC-containing metal complexes including silver, gold, copper, palladium and platinum as element has now been successfully studied on cancer cell lines.19-24
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Since the first report of platinum-NHC complexes as potential drug candidates in 2010 by Marinetti and co-workers,31,32 several groups have confirmed in vitro the cytotoxic effect of such platinum complexes.33-36 However, the development of these compounds is facing multiple challenges, among which the water solubility issue.37 Indeed, one usual limitation of NHC metal complexes is their weak solubility under biological conditions thus limiting their current use for in vivo experiments. We have previously reported several approaches for the post-functionalization of NHC-Pt complexes with the aim to introduce specific features that would improve their overall potential for in vivo experiments.34,38 In particular, the trans [(NHC)PtX2(pyridine)] (X = halide) complex in which pyridine can be easily exchanged by a nitrogen-based ligand was used to increase chemical diversity.33,39 The introduction of ethylenediamine ligand to the NHC-Pt fragment was found to improve the water solubility of the complexes. In this study, we reasoned that we could take advantage of this concept to generate multivalent cationic platinum complexes starting from linear polyethylenimine (PEI), a polymer widely used as a transfection agent (Figure 1).40-42
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Bioconjugate Chemistry
H3 N
H2 O N O Pt N O O H2 oxaliplatin
Cl Pt
H 3N
Cl
cisplatin
+
+ Me N C N Bn I Pt I
Me N C N Bn
Me N C N Bn I Pt NH2
H2 N
I
Pt
H 2N
N H
I OH 2a
H 2N
2b
H 2N
Me N C N + I Bn Pt NH HN
NH2
I
2c
I
NH2
N H n m
2PEI
Figure 1. Chemical structures of platinum complexes cisplatin, oxaliplatin, N-heterocyclic carbene-containing Pt complexes 2a-c and 2PEI.
Results and discussion Synthetic procedure and characterization The control monomeric NHC-Pt complexes 2a-c were synthesized as previously described by reacting trans [(NHC)PtX2(pyridine)] (NHC = 3-benzyl-1-imidazolilydene) 1 and the desired nitrogen-containing ligand (aminoethanol, ethylenediamine or diethylene triamine) (Figure 1).39 The multivalent 2PEI complexes were readily prepared using very similar conditions and linear PEI (25 kDa) as ligand as shown on Figure 2. The complex/polymer ratio was varied in order to obtain NHC-Pt-PEI conjugates that contain one Pt complex per 10, 20 or 30 ethylenediamine units (2PEI10, 2PEI20, 2PEI30, respectively), which correspond to ca. 60, 30 and 20 platinum atoms per PEI chain, respectively. Elemental analysis and 1H NMR on all
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compounds confirmed the final stoichiometry of the conjugates. Interestingly, all NHC-Pt complexes showed an improved hydrosolubility. Whereas stock solutions in DMSO are generally required to solubilize NHC-metal complexes, complexes 2a-c and 2PEI are routinely solubilized in ethanol at a concentration of 1 mM and then diluted in water for biological studies (Final concentration of ethanol at 10 µM = 0.5%). To further characterize the NHC-Pt-PEI conjugates prior to in vitro and in vivo experiments, we performed Cryo-electron microscopy experiments (cryo-EM) (Figure 3). The cryo-EM analysis revealed that 2PEI30 self-organizes into spherical particles. Most of these objects showed a diameter between 50 and 200 nm with a mean diameter around 150 nm (Figure 3, D).
Figure 2. Synthesis of NHC-Pt-PEI conjugate 2PEI30, prepared from trans [(NHC)PtX2(pyridine)] (NHC = 3-benzyl-1-imidazolilydene) 1 (EtOH, 55°C, 48h, quantitative).
The formation of these well-defined objects was not dependent on the concentration in the range 100 µM (Figure 3, A-B) to 500 µM (Figure 3, C). In contrast, very few objects were visualized when PEI alone was used as a control (Figure 3, E), with the exception of a few aggregates with no specific morphologies (Figure 3, F). The range of particle sizes determined by dynamic light scattering (DLS) was found to match that measured by cryo-EM.
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Figure 3 Cryo-EM observation of 2PEI30 and PEI. Low magnification of 2PEI30 (A) show a large number of spherical particles present at concentrations ranging from 100 µM (B) to 500 µM (C). D: measurements of the diameter of the particles observed at both concentrations showing that the majority of the spheres present a diameter between 50 and 200 nm. Images of PEI alone present almost no objects (E) except few aggregates (F). Scales bars: A-E: 1µm and B-C-F: 100 nm.
In vitro study The cytotoxicity of the complexes on cancer cells was investigated on a panel of 4 cancer cell lines and 1 non cancer cell line, namely, HCT116 (human colorectal adenocarcinoma), U87
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(human glioblastoma), PC3 (human prostate adenocarcinoma), TC1 (murine lung carcinoma), and a murine non cancer cell line, 3T3 (fibroblast). The toxic effects of NHC platinum were evaluated by measuring cell metabolic activity using 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxy-phenyl)-2-(4-sulfophe-nyl)-2H-tetrazolium (MTS) after 24 h of treatment. Cisplatin and oxaliplatin complexes were used as references in this study. The neutral NHC platinum complex containing the ethanolamine ligand 2a displayed cytotoxic activity, whereas the cationic complexes 2b and 2c were found to be completely inactive. The cytotoxic activity of 2a in this assay was found to be superior to cisplatin and in the same order of magnitude as oxaliplatin as shown in Table 1, top. Remarkably, the high molecular weight NHC-Pt-PEI conjugates 2PEI were the most potent complexes compounds in this series (Table 1, bottom, concentration values based on platinum). As shown in Figure 4a for human colorectal carcinoma cells HCT116, 2PEI conjugates were much more potent that PEI alone. Surprisingly their cytotoxic activities inversely correlate with the Pt/polymer unit ratio. A three-fold decrease in thePt/PEI unit ratio resulting in a ca. 10-fold increase in potency (IC50 ~ 21 µM for 2PEI10, IC50 ~ 7 µM for 2PEI20 and ~ 3 µM for 2PEI30) was observed. Finally, 2PEI30 ismuch more effective than oxaliplatin or cisplatin at the same platinum concentrations as shown in Figure 4b. The same conclusions can be drawn from cytotoxicity measurements on other cancer cell lines as shown in Table 1, bottom with a similar gain in activity observed upon increasing the PEI unit/Pt ratio from 10 to 30.
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Figure 4. Effects on HCT116 cell metabolic activity a) of NHC-Pt-PEI conjugates 2PEI10, 2PEI20, 2PEI30 and PEI; b) of oxaliplatin, cisplatin and 2PEI30. Results are expressed as mean+/SEM of at least 3 independent experiments (all concentrations are expressed as platinum concentration except for PEI).
Importantly, no change of in vitro activity was observed while keeping the NHC-Pt-PEI conjugate 2PEI30 in ethanol solution for 3 months (at 4°C). Finally, we did not see any change by UV-vis for the NHC-Pt-PEI conjugate under physiological condition after several days. Overall these results support a key role of linear PEI in amplifying the activity of NHC-Pt complexes, which may be rationalized in part by multivalency effects. Alternatively, linear PEI may also both serve to protect the metal from side reactions with undesirable molecules such as serum proteins, similar to what has already been shown for DNA in PEI/DNA polyplexes. A reason why 2PEI30 is the most active conjugate is perhaps related to the fact that
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some inherent properties (e.g. interaction with the lipid bilayer, buffering capacity over a broad pH range, endosomal release)43 of free PEI might be better preserved than in conjugates with more NHC-Pt units (2PEI10 and 2PEI30). Alternatively, 2PEI30 conjugate may be endowed with physicochemical properties, such as the ability to form well-defined spherical nanoparticules, revealed by cryo-EM studies which may facilitate endocytosis.
Cisplatin Oxaliplatin 2a
2b
2c
U87
>100
24±0.1
36±2
>100
>100
HCT116
>100
35.5±6
35±2
>100
>100
PC3
39±1.4
16±0.6
36±2
>100
>100
3T3
13±0.1
15±0.1
38±2.4
>100
>100
TC1
>100
27±2
28±3.5
>100
>100
PEIb
2PEI10c
2PEI20c
2PEI30c
U87
81±4
23±1
7±0.2
2.5±0.1
HCT116
60.5±3.8
21±1.8
6.5±0.1
2.8±0.1
PC3
47±4.2
17±1.7
27±1
1.8±0.1
3T3
>100
18±0.8
9±0.6
1.8±0.2
TC1
63.5±0.3
16±1.4
5±0.2
1.8±0.1
a
The IC50 values are determined by at least three independent assays. PEI polyethyleneimine 25 kDa. c Concentration expressed as platinum concentration. b
Table 1. In vitro cytotoxic activity (IC50 values in µM) of platinum complexes against several cell lines (after 24 h of incubation, stock solutions in EtOH for all complexes, stock solution in H2O for references).a
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To gain better understanding of the cell death mechanism and prior to in vivo investigation, we next investigated the induction of apoptosis by 2PEI30 of HCT116 cells by using nucleus staining with Hoechst 33342 or membrane staining using Annexin V. Our results indicate that NHC-Pt-PEI conjugate 2PEI30 at concentrations > 20µM induced a high level of apoptosis in comparison with oxaliplatin at the same concentrations (Figure S5).
In vivo study In view of these very encouraging results, in vivo experiments were conducted with the NHCPt-PEI conjugate 2PEI30 to evaluate its antitumoral activity in an HCT116 (human colorectal cell line) tumor xenograft mouse model. While 2PEI30 was easily formulated (stock solution at 10 mg mL-1 in absolute ethanol and then diluted in NaCl 0.9% containing 10% of ethanol) for in vivo experiments, the low hydrosolubility of 2a precluded its use in vivo. Thus, PEI polymer may serve as carrier for in vivo studies as lipophilic Pt complexes like 2a are not suitable for in vivo administration without formulation. In contrast to mice treated with vehicle control only, significant tumor volume inhibition (by 80%, p
