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Synthesis of Highly Fluorescent Copper Clusters Using Living Polymer Chains as Combined Reducing Agents and Ligands Markus J Barthel, Ilaria Angeloni, Alessia Petrelli, Tommaso Avellini, Alice Scarpellini, Giovanni Bertoni, Andrea Armirotti, Iwan Moreels, and Teresa Pellegrino ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b04270 • Publication Date (Web): 29 Oct 2015 Downloaded from http://pubs.acs.org on November 2, 2015
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“Synthesis of Highly Fluorescent Copper Clusters Using Living Polymer Chains as Combined Reducing Agents and Ligands” Markus J. Barthel1, Ilaria Angeloni1,2, Alessia Petrelli1, Tommaso Avellini1, Alice Scarpellini1, Giovanni Bertoni1,3, Andrea Armirotti,1 Iwan Moreels1, Teresa Pellegrino1,* 1
Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova, Italy
2
Dipartimento di Chimica, Università di Genova, Via Dodecaneso 33, 16146 Genova, Italy
3
IMEM-CNR, Parco Area delle Scienze 37/A, 43124 Parma, Italy
*Corresponding author:
[email protected] TOC
Abstract We present the synthesis of colloidally stable ultra-small (diameter of 1.5 ± 0.6 nm) and fluorescent copper clusters (Cu-clusters) exhibiting outstanding quantum efficiencies (up to 67% in THF and approx. 30% in water). For this purpose, an amphiphilic block copolymer poly(ethylene glycol)1 ACS Paragon Plus Environment
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block-poly(propylene sulfide) (MPEG-b-PPS) was synthesized by living anionic ring-opening polymerization. When CuBr is mixed with the living polymer chains in THF, the formation of Cuclusters is detected by the appearance of the fluorescence. The cluster growth is quenched by the addition of water, followed by THF removal. The structural features of the MPEG-b-PPS copolymer control the cluster formation and the stabilization: the poly(propylene sulfide) segment acts as coordinating and reducing agent for the copper ions in THF, and imparts a hydrophobic character. This hydrophobic block protects the Cu-clusters from water exposure thus allowing to obtain a stable emission in water. The PEG segment instead, provides the hydrophilicity, rendering the Cu-clusters water soluble. To obtain fluorescent and stable Cu-clusters, exhibiting outstanding quantum efficiencies, the removal of the excess of free polymer and copper salt was crucial. The Cu-clusters are also colloidally and optically stable in physiological media and showed bright fluorescence even when taken up by HeLa cells, being non-cytotoxic when administered at a Cu dose between 10 nM and 1.6 µM. Given the very small size of the Cu-clusters, localization and fluorescent staining of cell nucleus is achieved, as demonstrated by confocal cell imaging performed at different Cu-cluster doses and at different incubation temperatures.
Keywords Copper clusters, nanoparticles, anionic polymerization, PEG, propylene sulfide, photoluminescence, cell uptake
Next to classical metallic nanoparticles (NPs), such as gold and silver NPs,1-7 copper NPs represent a new class of metallic NPs with a multitude of fields of application. One commonly exploited property of copper is the high electrical conductivity. Copper NPs can be used for the production of conductive inks used for fabrication, via inkjet processes, of micrometre scale patterns.8 Alternatively, the catalytic activity of copper NPs is also relevant in some organic reactions. In 2 ACS Paragon Plus Environment
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particular, the catalyzation of C-N, C-C, C-O, C-S and C-P bond-forming reactions and hydroxylation of organic compounds mediated by Cu NPs were recently reported.9, 10 In analogy to very small Au and Ag NPs, if the size of copper NPs is reduced to less than circa 3 nm, the copper clusters (Cu-clusters) become fluorescent. Their fluorescence can then be used, e.g., for the detection and photocatalytic elimination of lead ions.11, 12 Indeed, the fluorescence of aqueous Cuclusters is rapidly quenched if lead ions in solution are adsorbed on the particle surface. Upon UV irradiation, the Cu-clusters reduce the lead ions to metallic lead, which could be then eliminated from the solution. Additionally, the formation of fluorescent Cu-clusters can be used for the detection of single nucleotide polymorphisms.13 In this case, copper ions are reduced by ascorbic acid in the presence of DNA duplexes. The resulting fluorescence is dependent on the surrounding base pairs. In the cases of mismatches a higher fluorescence can be observed. Besides DNA diagnosis, the fluorescence of the clusters can be exploited for cell imaging. To this aim Cu-clusters should exhibit, at the same time, high fluorescence under physiological conditions and at body temperature, and should be also colloidally stable. Even if Cu-clusters/NPs represent a promising candidate for large scale applications (the material costs of the precursor for the synthesis of Cu-clusters or NPs is substantially lower with respect to gold or silver precursors), the controlled synthesis of small Cu-clusters is still challenging. Classically, for the synthesis of Cu-clusters/NPs, the reduction of Cu(II) ions by sodium borohydride, hydrazine or ascorbic acid is employed. This reaction is often performed in the presence
of
stabilizing
agents
carrying
thiol
groups,
such
as
DNA,
proteins,
mercaptopropylpyrimidine, tetrabutylammonium nitrate or dodecanethiol.14-18 Alternatively, also the controlled synthesis of Cu-clusters/NPs in ionic liquids,19 by photo-irradiation,20 or by electrochemical processes21 is possible. Despite the wide range of synthesis routes, several synthesized Cu-clusters/NPs revealed a lack of stability against oxidation at the human body temperature with adverse effects on their fluorescence properties.22 Moreover, the stabilizing 3 ACS Paragon Plus Environment
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ligands including mercaptopropylpyrimidine, tetrabutylammonium nitrate or dodecanethiol, used in some of the preparation methods need to be replaced after the synthesis with a hydrophilic and more biocompatible ligand shell. A common feature of most of the above described Cu-clusters is their absorption in the UV. However, in case of cell imaging application, upon excitation in the UV, the strong auto-fluorescence of the tissue under UV light results in a lower signal to noise ratio with a reduced quality of the
fluorescent imaging of the Cu-clusters.23 Therefore, the biological
applications of Cu-clusters are up to now limited. Some attempts to grow Cu-clusters directly in water were recently reported. In this regard, colloidally stable aqueous fluorescent copper NPs stabilized by a PEG-containing ligand were recently reported.24 A lipoic acid functionalized PEG750 polymer was used as ligand for the synthesis of fluorescent Cu-clusters and NaBH4 was used as reducing agent for the copper ions. A drawback of the reaction however is the long reaction time (48 h) and the high reaction temperature (100 °C) with a final quantum efficiency (QE) of the resulting Cu-clusters of 3.6%. In another work, glutathione (GSH) stabilized Cu-clusters could be synthesized in a one-pot reaction in aqueous solution.25, 26 The GSH ligands worked as reducing agent for the Cu(II) salt and also as stabilizer for the formed fluorescent Cu-cluster of few nanometers in size. Interestingly, the GSHCu-clusters showed a heat dependent quenching of the fluorescence which the authors have proposed as an intracellular thermometer.26 Very recently, Ghosh et. al. have described another aqueous synthesis of Cu-clusters/hydrogel with an overall diameter of 300 nm, using the reduction of copper ions incorporated into a polymeric matrix of PVP and PVA by ascorbic acid and dihydrolipoic acid.27 The Cu-hydrogels were used for imaging the cell uptake and for the delivery of cis-platinum as anticancer agent. Additionally, the presence of Cu promotes the reactive oxygen species (ROS) production with synergetic cytotoxicity effects on top of that of cis-platinum. Inspired by these works, in order to control the synthesis of ultra-small Cu-clusters, we choose a diblock copolymer, i.e. poly(ethylene glycol)-block-poly(propylene sulfide) (MPEG-b-PPS), having 4 ACS Paragon Plus Environment
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multifunctional subunits which merge the advantages of the previous ligands in one single polymeric ligand. The PEG segment ensures water solubility, good biocompatibility and a stealth effect against protein adsorption to the Cu-clusters.28, 29 The thiolate end-group of the PPS segment can be used to coordinate the Cu ions and to reduce a copper (I) salt (in our case CuBr) to Cu(0) and form Cu-clusters. Furthermore, due to the hydrophobic character of the poly(propylene sulfide) segment the Cu-clusters can be protected against water contact and oxidation.30-32 For the polymer preparation we exploited the living anionic polymerization, as it is a wellestablished method for the synthesis of PEG or functional epoxides, and provides a high stability of the negative charge at the chain-end due to the absence of termination and coupling reactions.33-36 This negative charge can be used for the stabilization and reduction of metal. Among them, PEG thiolates carrying a negative charged S- at the chain-end are reported as initiators for the living anionic ring-opening polymerization of propylene sulfide.37 The formed poly(propylene sulfide) segment has a highly hydrophobic character. In the following, we present a novel method for the synthesis of stable and highly fluorescent Cuclusters with an outstanding quantum efficiency. The process relies on the synthesis of living amphiphilic polymer chains of MPEG-b-PPS and the subsequent mixing with CuBr under inert conditions in THF. The cluster formation is quenched by pouring the reaction mixture in water followed by the evaporation of the THF.
Results and Discussion Polymer synthesis To prepare poly(ethylene glycol)-block-poly(propylene sulfide) (MPEG-b-PPS) as an amphiphilic di-block copolymer applied as combined stabilizing ligand and reducing agent, living anionic ringopening polymerization was used following a procedure reported in literature.37 Briefly, α-methoxyω-thioacetate-poly(ethylene glycol) (MPEG-SAc) is used as polymeric precursor, obtained by a 5 ACS Paragon Plus Environment
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two-step reaction starting from the tosylation of commercial methoxy-poly(ethylene glycol) (MPEG, Mn = 2,000 g/mol) and subsequent reaction with potassium thioacetate (see supporting information, SI, Scheme S1). In anhydrous THF and under inert atmosphere, the MPEG-SAc precursor was activated by sodium methanolate, resulting in cleavage of the acetyl group of MPEG-SAc and the formation of the living MPEG-thiolate species. Subsequently, the propylene sulfide monomer (aiming a degree of polymerization (DP) of 38) was added for the formation of the PPS block as depicted in Scheme 1. The full conversion of the MPEG-SAc precursor to the MPEG-b-PPS block copolymer was ensured by size-exclusion chromatography (SEC) measurements (Figure 1e). The detailed procedure is reported in the experimental part. No purification step for the polymer was applied and the crude mixture of the reaction was directly used in the next step for the growth of the Cu-clusters.
Scheme 1: Schematic representation of the living anionic polymerization of propylene sulfide to form living α-methoxy-poly(ethylene glycol)-block-poly(propylene sulfide) (MPEG-b-PPS) chains and subsequent reaction with Cu(I) ions for the formation of Cu-cluster.
Synthesis of fluorescent Cu-clusters In a typical Cu-cluster synthesis, after the formation of the MPEG-b-PPS block copolymer, the solution containing the living polymer chains (0.2 mmol of polymer / 20 mL of THF), carrying a stable negative charge at the chain-end, was transferred into a flask containing CuBr as metal precursor (0.45 mmol, 64 mg). The mixture was allowed to stir for 30 min at room temperature (RT) under nitrogen atmosphere. A colour change from colourless to a pale yellow could be 6 ACS Paragon Plus Environment
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observed within the first 5 min. If the reaction mixture was illuminated with a UV lamp (wavelength 365 nm) a blue-green fluorescence was observed after approximately 1 min reaction time, changing to yellow-orange after 30 min (Figure S1). This phenomenon is commonly associated in literature with the formation of small Cu-clusters.18 The growth of the Cu-NPs was subsequently quenched by pouring the THF reaction mixture into 40 mL of water (we always used the double amount of water with respect to THF). The mixture was poured into water swiftly, to ensure that the hydrophobic PPS segment could immediately form a protective layer on the Cu surface and prevent water contact (thus avoiding possible oxidation of the formed Cu-clusters). The THF was evaporated under nitrogen flow for 30 min resulting in a stable and clear aqueous dispersion of NPs. Using this approach a yellow dispersion of the crude sample in water (still containing an excess of the polymeric ligand) was obtained (Figure S2, left). If the solution was irradiated with a UV lamp (wavelength 365 nm) a strong yellow emission could be observed (Figure S2, right). To test if the reaction time has an influence on the resulting Cu-cluster size, the contact time of the living chains with CuBr was reduced to only 1 min (i.e., after 1 min the reaction was quenched by water addition). As shown in Figure S3 a strong blue shift of the emission signal was observed, indicating the formation of smaller particles. Nevertheless, the latter results were not completely reproducible, as most probably at very short times small clusters can be very reactive. To ensure reliable results we focused for the following experiments on a reaction time of 30 min. To further confirm that the PPS segment is crucial for the Cu-cluster synthesis, a reference reaction was performed using living anionic MPEG-S- chains to reduce CuBr (Scheme S2). In contrast to the stable solution of Cu-clusters obtained by the method described above, in this case an aggregation of the formed Cu-clusters was observed as soon as the reaction mixture was poured in water and the THF was removed (Figure S4a and b). This process led to a rapid macroscopic precipitation of the material (Figure S4c) confirming, therefore, the crucial role of the stabilizing PPS block. 7 ACS Paragon Plus Environment
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To remove the excess of free polymer, which in water forms micellar nanostructures with a diameter of 18 nm (Figure S5), a simple washing of the sample by, for instance, a centrifuge filter was not possible, since the free polymeric micelles as well as the Cu-clusters cannot pass the membrane of the filter and cannot be separated. Also ultracentrifugation or (size-exclusion) column chromatography did not yield pure Cu-cluster fractions. Therefore, to isolate the Cu-clusters from the polymeric excess, a new purification protocol had to be developed. The crude sample in water was concentrated by a centrifuge filter (cut-off 30,000 g/mol) to a volume of approx. 15 mL. Afterwards, the mixture was transferred in a dialysis membrane (molar mass cut-off of 3,500 g/mol, spectrum labs regenerated cellulose (RC) membrane) and the sample was dialyzed for 3 d against THF (the solvent was changed after 6 h and subsequently every day while all the THF fractions were kept). The formed Cu-clusters were small enough to pass through the dialysis membrane (Figure 1a and b). The excess of free polymer and copper salt remained inside the dialysis bag and did not show fluorescence (Figure 1c). All THF fractions from the dialysis, except the first one taken after 6 h which contained the water from the crude sample, were combined and concentrated under reduced pressure to a final volume of 4 mL. A clear, colourless solution in THF could be obtained (Figure 1d) which showed fluorescence under the UV lamp (excitation wavelength 365 nm), suggesting that the Cu-clusters were transferred in THF (Figure 2d). Moreover, since the solubility of the copper salt in THF is lower than in water, the excess of copper precipitated inside the membrane and did not cross the membrane pores (Figure 1b).
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Figure 1: Purification process of the crude Cu-cluster sample from the excess of polymeric ligand and CuBr by dialysis against THF using a RC membrane (cut-off 3,500 g/mol) at t=0 (a), after 4 d (b). (c) Residue within the dialysis membrane. (d) Concentrated THF fractions collected outside the membrane. (e)SEC characterization (solvent: THF with a flowrate of 1 mL/min): Elution trace of the polymeric residue remaining into the dialysis bag (black line) containing dimerized MPEG-b-PPS and the excess of unreacted MPEG-b-PPS ligand, the purified Cu-clusters recovered outside the membrane bag (red line), and the polymeric MPEG-SAc precursor.
SEC was used to verify the dialysis separation by comparison of the solution fraction collected from the dialysis bag with that corresponding to the dialysis solvent, the THF fraction (Figure 1e). The polymeric residue within the dialysis bag, corresponds to polymer chains which did not act as ligands for the cluster representing the unreacted polymer, is characterized by a bimodal molar mass distribution (Figure 1e, black line). The signal at lower elution volume showed the double molar mass of the signal at higher elution volume, thus likely represents dimerized polymer chains (MPEG-b-PPS-S-S-PPS-b-MPEG). Possibly, as the unreacted thiolate of the single MPEG-b-PPS chains are sensitive to protic impurities, upon the addition of water used to quench the Cu-cluster formation, the thiol end-groups can dimerize leading to the formation of S-S bonds between MPEG9 ACS Paragon Plus Environment
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b-PPS- chains. After purification, the concentrated THF fractions collected outside the membrane
bag, reveal a monomodal mass distribution at lower molar mass in comparison to the free polymer signal found within the dialysis bag, confirming the successful purification and removal of the excess of the free polymer (Figure 1e, red line). Overall these data suggest that the free polymer with a molar mass of approx. 4,800 g/mol is too large to pass the dialysis membrane (having molecular mass cut-off of 3,500 g/mol). By comparison of the elution volume in SEC, a corresponding higher elution volume is recorded for the purified Cu-cluster with respect to that of the free polymeric residue. Also, the Cu-clusters passed the pores of the dialysis membrane while the polymer residue did not. This suggest that due to the high number of S atoms carrying free electron pairs and having high affinity for the Cu, the polymeric PPS block enwraps the Cu-cluster very tightly resulting in a smaller hydrodynamic radius (more coiled configuration) compared to that of the free polymer (more stretched configuration). It is important to mention that in both samples no signal was detected at the elution time of the MPEG-SAc precursor (Figure 1e, blue line). This proves the complete activation of the precursor during the polymerization reaction, and the complete transformation of MPEG-SAc into a MPEG-b-PPS block copolymer. To compare the optical properties of the crude and purified Cu-clusters, UV-Vis absorption and emission measurements were performed (Figure 2a and b). The absorption spectrum of the crude Cu-clusters in water (not purified from the excess of free polymer) shows no features in the visible region (Figure 2a, blue line). Photoluminescence (PL) measurements when exciting the sample at a wavelength of 445 nm show a broad emission signal from approx. 550 to 750 nm, with increasing intensity at higher wavelengths. The corresponding photoluminescence emission (PLE) spectrum in the range from 360 to 735 nm was collected by measuring the emission intensity at 750 nm (Figure 2b, dotted blue line). Consistent with the absorption measurements, a featureless signal was
observed, with increasing intensity toward lower wavelengths. Furthermore, the PL decay trace was measured on crude Cu-Clusters, revealing a lifetime of around 1.5 µs, and the PL QE fell below 1% 10 ACS Paragon Plus Environment
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for this sample (Figure S6a). These data suggest that before purification, clustering of the Cu NPs, possibly in micelles formed by the excess of polymer, leads to efficient separation of photo-excited charges across different Cu-Clusters, leading to long lifetimes and low PL QE due to competition with non-radiative trapping.
Figure 2: (a) Absorption spectra of the “crude” Cu-clusters in water (blue line), and of the “pure” Cuclusters in THF (red line) and in water (green line) respectively, after removal of the excess of polymer. While the crude Cu-clusters are featureless in the visible, absorption transitions are present in the pure sample in THF at 390, 500 and 530 nm which can still be identified when the sample is transferred in water. (b) Normalized PL spectra of the three samples (solid lines with the same colour code as for absorption spectra, excitation wavelengths: 445 nm for the crude sample, 530 nm for the pure samples). Corresponding PLE spectra (dashed lines) were measured observing the emission at 750 nm for the crude sample and at the maximum of the PL peak for the pure samples. (c) Representative images of crude Cu-clusters in water under daylight (left) and upon UV irradiation at 365 nm (right). (d) Pure Cu-clusters in THF under daylight (left), and upon UV irradiation at 365 nm (middle) and upon irradiation with a torch lamp (right). (e) Pure Cu-clusters in water under daylight (left) and upon UV irradiation at 365 nm (right).
The spectroscopic measurements were repeated on the purified Cu-Clusters in THF (Figure 2a and b, red lines). In this case, the absorption spectrum shows clear transitions at 390, 500 and 530 nm,
which were not observed in the crude sample in water. Some studies in the literature state that no clear absorption features should be present for small Cu-clusters.24, 27 On the other hand very small 11 ACS Paragon Plus Environment
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Cu-clusters (
