M.
Rodrigues
a,
L.
Russo
a,
E.
Aguiló
b,
L.
Rodríguez
b,
I.
Ott
c and
L.
Pérez-García‡
*a
aDepartament de Farmacologia i Química Terapèutica and Institut de Nanociència i Nanotecnología UB (IN2UB), Universitat de Barcelona, Avda. Joan XXIII s/n, 08028 Barcelona, Spain. E-mail: mlperez@ub.edu
bDepartament de Química Inorgànica, Universitat de Barcelona, C/Martí i Franquès 1-11, 08028 Barcelona, Spain
cInstitute of Medicinal and Pharmaceutical Chemistry, Technische Universität Braunschweig, Beethovenstr. 55, 38106 Braunschweig, Germany
First published on 22nd December 2015
A gold(I) N-heterocyclic carbene 4 from a bis-imidazolium-amphiphile was synthesized and characterized. The cytotoxicity against HT-29 colon carcinoma and MDA-MB-231 breast adenocarcinoma cells was assessed for the NHC complex 4, the imidazolium salt precursor 2, and its methyl analogue 3, indicating that compounds 2–4 are promising cytotoxic agents. Furthermore, the ability of these compounds to be associated with gold nanoparticles was also explored, in order to develop an anticancer drug delivery system. The free ligands displayed more activity when compared with the ligands immobilized on the gold nanoparticles. The synthesized gold particles incorporating the bis-imidazolium salts either 2 or 3 showed monodisperse spherical shape with sizes of approximately 5 nm.
Thioredoxin reductase (TrxR) is an important and ubiquitous enzyme critically involved in the regulation of intracellular metabolism. The thioredoxin/thioredoxin reductase system plays an important role in the redox state of the cells, besides being involved in other cell functions such as cell proliferation, transcription factor regulation or apoptosis.7 Inhibition of this system causes oxidized thioredoxin to accumulate in cells, promoting apoptosis, thus making thioredoxin reductase a good target for cancer therapy.8,9 In this context, increasing interest has grown towards the development of inhibitors based on lipophilic Au(I) complexes.10 These complexes are a very promising class of non-platinum based antitumor agents, and among them neutral and cationic complexes with N-heterocyclic carbenes (NHCs) as ligands show a remarkable strong anti-cancer effect through the inhibition of Trx/TrxR reductase system.10 Recent results indicate that TrxR inhibition in combination with antimitochondrial effects are key properties that determine the bioactivity of gold NHC complexes. This therapeutic strategy exploits the negative mitochondrial membrane potentials to selectively concentrate delocalised lipophilic cations, such as Au(I) complexes, within the organelle. In addition, the gold complex provides the compound the ability to selectively coordinate the enzyme's functional selenocysteine, increasing its anti-tumour activity.10
N-Heterocyclic carbenes (NHC) metal complexes11,12 are known and studied as potential anticancer metallodrugs because, compared with traditional gold complexes such as auranofin, they show an enhanced stability of the coordinative bond with the metal atom and therefore they likely do not undergo fast metabolization before reaching their enzymatic target.13 Despite the similarities with the well-known phosphine–gold complexes,4 NHC ligands represent a better alternative due to their stability, as well as the ease with which it can be functionalized in order to vary their lipophilicity. Nowadays, different groups concentrate their investigation on developing novel gold NHC complexes with anti-cancer properties. Schuh et al. successfully synthesized a family of substituted imidazole and benzimidazole asymmetric Au(I) linear NHCs, evaluating both their antiproliferative effects on human ovarian cancer cell lines and their effective inhibition of TrxR.14 Within the same type of NHC–Au–L compounds, Rubbiani et al. compared the effect of different ligand L (L = –Cl, –NHC, or –PPh3) on TrxR inhibition and antimitochondrial action,15 showing how both the cationic character and the modulation of the stability of the coordinative bond of the complexes are key features for their cytotoxicity. Liu et al. also provides a further example of a diarylimidazole-based gold NHCs, in which a correlation between complexes' biological activity and both the inactive, non-NHC ligand and the aromatic substituents can be established.16 We have also described the synthesis of Au(I) N-heterocyclic monocarbenes and dicarbenes derived from imidazolium salts, their biological activity and TrxR inhibition ability.17
Moreover, recent works provide evidence of the wide range of functionalities that can be introduced in this class of heterocyclic compounds in order to further investigate their antitumor activity: Pratesi et al.18 explored the inhibition mechanism of TrxR by successfully coupling three different Au(I)–NHC complexes with a synthetic dodecapeptide containing a selenocysteine group, confirming the direct coordination of this thiol-residue with the gold atom. Citta et al. took advantage of the ease of modification of NHCs for providing their complexes with a fluorescent anthracene unit. This functionalization not only does not affect the gold compound's antiproliferative effect, but also allows the study of its distribution in vivo.19
In our group, a class of gemini-type imidazolium surfactants has been extensively studied because of its anion recognition properties20,21 as well as its capability of stabilizing gold nanoparticles (AuNPs) synthesized in a biphasic system.20,22 The amphiphilic bis-imidazolium compounds play a double role in the synthesis, acting as transfer agents and stabilizers. Furthermore, their anion recognition ability allowed the AuNPs to incorporate successfully a model anionic drug and release it in a sustained manner. Additionally, by changing the synthetic method, we were able to produce amphiphiles bilayer-coated water-soluble AuNPs that could load and deliver piroxicam, an anti-inflammatory drug with poor water solubility.23 Therefore, these imidazolium-based molecules are promising materials for biomedical applications.24 It would be interesting to go one step further and add a novel role to these molecules by introducing a metal complex with gold moiety, thus achieving AuNPs that would have not only the ability to carry drugs, but also present biological activity due to the presence of the N-heterocyclic carbenes (NHC) metal complex. The strategy to achieve this goal would include synthesizing the NHC–Au(I) complex, but also to take advantage of the versatility of these molecules, a propargyloxy group was introduced in order to allow the formation of a phosphine–Au(I)-alkynyl derivative.
Thus, in this study we develop the synthesis of novel bis-imidazolium Au(I) carbene complexes as multifunctional ligands for the synthesis of AuNPs with potential anticancer activity, and we report: (a) the synthesis and structural characterization of a bis-imidazolium gold(I) complex, (b) the synthesis and morphological characterization of AuNPs, bearing different kinds of bis-imidazolium amphiphilic ligands as stabilizing agents, and (c) the evaluation of the biological activity of both the ligands and AuNPs.
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Scheme 1 Synthesis of imidazolium-based compounds 2 and 3, formation of the carbene complex 4 and attempt of the formation of complex 5 with phosphine–Au(I)-alkynyl derivative. |
Two parallel strategies were then followed in order to obtain the imidazolin-2-ylidene-Au(I) complex 4 (Scheme 1). The aim was to direct selectively the ligand substitution reaction of the gold precursor towards one precise functional group by varying the reaction conditions and reagents. In the first strategy, by following a general procedure described in literature,26 we targeted the acidic proton of the imidazolium rings, in order to form a neutral heterocyclic carbene (NHC) as a coordinating reactive site. Compound 4 was successfully synthesized by the reaction of [AuCl(SMe2)] with 2, deprotonated in situ with lithium bis(trimethylsilyl)amide (LiHMDS) in anhydrous acetonitrile.11 This procedure provided selective conditions for the synthesis of the Au(I)-carbene complex 4, obtained as a colourless solid by recrystallization from acetone–hexane or ethanol–diethylether with a 40% yield. The second strategy focused on targeting the terminal alkyne of the propargylic ether by deprotonating it in situ with potassium hydroxide, promoting the coordination with a gold-1,3,5-triaza-7-phosphaadamantane-chloride complex [AuCl(PTA)].27 Despite the relatively mild basic environment, deprotonation was not obtained selectively on the propargylic ether of compound 2 but a competition with the imidazolium acidic proton was observed. Various obstacles during purification were encountered, probably due to the lack of regioselectivity, thus an univocal structure was not identified. To solve this inconvenience, the same reaction was carried out on the methylated bis-imidazolium ligand 3, in an attempt to obtain the complex 5 incorporating a phosphine–Au(I)–alkyinyl derivative. However due to the tendency towards aggregation caused by the bulky PTA ligand, the desired gold complex could not be isolated in its pure form and consequently was not successfully characterized.
Compounds 2, 3 and 4 were characterized by 1H NMR spectroscopy. The corresponding spectroscopy data is summarized in Table 1.
While both the imidazolium dications maintain similar chemical shifts for their analogous groups (for instance the terminal alkyne proton peak at 2.64 ppm and 2.57 ppm respectively), the imidazolium substituent R in position 2 was indicative for distinguishing between the two compounds. The formation of Au(I) carbene complex 4 was also determined by 1H NMR spectroscopy (Table 1) by following the disappearance of the imidazolium acidic proton signal at 10.44 ppm, which might indicate the coordinative bond formation between the imidazolium dicarbene and the gold atom. The regioselectivity of the reaction was proved since Au(I) compound spectra presented always the peak corresponding to the alkynyl terminal proton at 3.53 ppm, with a Δδ = +0.89 ppm compared to the starting material.
The structures of compounds 2 and 3 were also confirmed by Electrospray Ionization Mass Spectrometry (ESI-MS) with both presenting a main characteristic fragmentation ion corresponding to the dication resulting of the loss of the two bromide counterions [(M − 2Br)/2]2+ (Table 2 and ESI, Fig. S1 and S2†).
MWb (g mol−1) | Ionsa (m/z) | |||
---|---|---|---|---|
[(M − 2Br)/2]2+ | [M − Br]+ | [M–Br–CH2CCH−]+ | ||
a Ions, m/z ratio relative abundance (%). b Molecular weight. c ESI-MS. d MALDI-TOF-MS with matrix DHB. e MALDI-TOF-MS without matrix. | ||||
2 c | 959.1 | 399.4 (100%) | ||
3 c | 987.2 | 413.4 (100%) | ||
4 d | 1074.2 | 993.8 (100%) | 955.7 (25%) | |
4 e | 993.7 (75%) | 955.7 (100%) |
Further confirmation of Au(I) carbene 4 was provided through Matrix Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) analysis which showed, in the case of DHB-supported ionization, two main peaks, the most abundant peak corresponding to bromide loss (M − Br)+ at 993.80 m/z, in accordance with previously described analogues,20 and the second peak from the simultaneous bromide and propargyl loss (M − Br − CH2CCH)+ at 955.70 m/z (see ESI, Fig. S3†). The measurement was also performed without polymer matrix and the same main characteristic signals were observed, together with peaks related to the gradual fragmentation of the alkyl chains (see ESI, Fig. S4†). There were however some differences observed in the abundance of the obtained peaks: when the analysis was performed without matrix, the most abundant peak corresponded to the loss of bromide and propargyl, whereas in the case of the analysis performed with matrix, the abundance is much lower (25%), and the peak corresponding to the molecule with no loss of bromide is also identified with low abundance (10%).
Following the previously reported procedure, the nanoparticle synthesis was initially carried out successfully using the free bis-imidazolium ligands 2 and 3 through the modified Brust–Schiffrin method. The AuNPs formation was monitored by UV-visible absorption spectroscopy, following the characteristic Surface Plasmon Resonance (SPR) band at ca. 520 nm (Fig. 1). The samples were further identified as 2·AuNP and 3·AuNP, respectively. After washing, the absorption spectrum was compared to the free ligand, whose peaks at around λ = 280 nm are clearly recognizable in the nanoparticles solution, confirming its presence as stabilizer.
Additionally, compound 2 and the corresponding 2·AuNP sample were analysed by Infrared spectroscopy (IR) and it was possible to identify the peaks of the ligand 2 on the AuNPs (see ESI, Fig. S5 and S6†). The peaks corresponding to the C–H bond in the imidazolium ring can be found around 3100 cm−1 (being less visible in the 2·AuNP spectrum). Two pronounced peaks, corresponding to the CH2 from the alkyl chains are found at 2850 and 2920 cm−1, around 2100 cm−1 a peak assigned to the CC is found (also less pronounced in the 2·AuNP spectrum). Between 1560 and 1650 cm−1 three peaks, that can be assigned to the C
C and C
N from the imidazolium moieties, are found. Generally, the peaks of imidazolium salts are less intense, especially the ones from the functional groups which are somehow interacting with the metallic gold, such as the phenyl ring and the alkine.
The synthesized AuNPs were observed by High Resolution Transmission Electron Microscopy (HRTEM) to study their morphology and size. The obtained micrographs can be seen in Fig. 2 and the size distribution can be observed in the corresponding included histograms.
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Fig. 2 HRTEM micrographs of 2·AuNP with (a) 30![]() ![]() ![]() ![]() |
It is possible to identify relatively monodisperse spherical particles with average diameter of 4.8 ± 1.1 nm and 4.2 ± 1.0 nm for 2·AuNP and 3·AuNP respectively.
By comparing the 2·AuNP and 3·AuNP nanoparticles, formed with either imidazolium ligand 2 or 3, both TEM images and UV-vis absorption spectroscopy did not provide any evidence of relevant morphological difference, suggesting that the introduction of a methyl chain in the imidazolium ring did not affect significantly the ability of the ligand 3 to stabilize AuNPs.
X-ray photoelectron spectroscopy (XPS) showed the presence of two peaks at 84.2 eV and 87.8 eV (in the case of 2·AuNP) and 85.2 eV and 88.8 eV (in the case of 3·AuNP) corresponding to Au4f7/2 and Au4f5/2 respectively. The peak position and the distance between the two peaks (3.7 eV) is consistent with gold in its reduced form Au(0) (see ESI, Fig. S7†) and is in agreement with previously described bis-imidazolium coated nanoparticles.20 Furthermore, the presence of minor peaks corresponding to Au(I), that can be found with a shift of 1–2 eV with respect to the Au(0) peaks,28–30 were not identified during the curve fit of the XPS spectra, suggesting that no carbene species were formed in situ during the AuNP synthesis, and that the interaction between the ligand and the gold surface is not mediated by carbenes.
The AuNPs were also analysed by X-ray diffraction (XRD) to determine the phase composition. Four Bragg peaks could be identified in the gold XRD pattern, indicating a face-centered cubic (fcc) structure, with peaks at 37°, 44°, 65° and 78°, corresponding respectively to (111), (200), (220) and (311) planes (see ESI, Fig. S8†).
Thermogravimetric analysis was performed on 2·AuNP. The data obtained for the amount of ligand present in the sample, together with size data obtained from size measurements, allowed the calculation of the number of ligands present in the AuNPs (see ESI, Fig. S9 and Table S1†). It was possible to determine that the AuNPs sample has approximately 24 ligands per nm2. This value is in agreement with previous described AuNPs with similar ligands, that present around 28–30 ligands per nm2.22,23 This data also allows to determine the amount of ligands present per gold nanoparticle, thus allowing to calculate the approximate molecular weight of the whole nanostructure.
Following these results, experiments were made to synthesize the Au(I)-complex directly on the formed 2·AuNP nanoparticles, with compound 2 as stabilizing agent. The goal was to obtain the advantages of the gold nanoparticle as drug carrier, and at the same time have a ligand with anticancer activity. To perform the synthesis of the NHC metal complex, the same procedure described in Scheme 1 was attempted, but the basic medium caused the colloid to precipitate, and this approach was discarded.
From the above values it can be observed that the free compounds do not follow a trend in the two studied cell lines as the observed IC50 values are within a rather narrow range (6–13 μM). For complex 4, due to limited solubility this compound could not be administered at concentrations higher than 10 μM, and it was found that the IC50 against HT-29 cells is higher than the maximum concentration tested.
The nanoparticles 2·AuNP and 3·AuNP were both applied at maximum dosages of 0.0044 μM and did not display cytotoxicity at this concentration. Using the data from the TGA analysis, it is possible to determine the molecular weight of the AuNP and to determine the amount of ligand present per mole of AuNP. The calculations made show that to the applied concentration of 0.0044 μM of AuNP corresponds a concentration of 16 μM of ligand. This value is similar to the IC50 found for the free compounds, which means that when bound to the AuNPs, the ligands do not present the same toxic effect. However, since this was the maximum concentration tested, it was not possible to determine precisely the IC50 value of the ligand bound to the AuNPs.
These findings are in agreement with the results obtained with a previously described analogue,20 also studied free and conjugated to AuNPs, where we found a similar outcome. The IC50 value (determined in Caco-2 cells) is similar to the one found for these compounds, but the corresponding AuNPs cytotoxicity was also higher than the maximum tested concentration. Therefore, these results suggest that the ligands alone present higher cytotoxicity, because they are more available for interaction with the cells.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra21621d |
‡ Present address: School of Pharmacy, The University of Nottingham, University Park, Nottingham NG72RD, England, UK. |
This journal is © The Royal Society of Chemistry 2016 |