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We synthesized green-emitting platinum (Pt) nanoclusters (excitation: 460 nm, emission: 520 nm) by reducing Pt ions from pre-equilibrated Pt/fourth-generation poly(amidoamine) dendrimers (PAMAM (G4-OH)) complexes with a mild reductant. The structural characteristics of the resulting Pt nanoclusters, Pt8L8 (L = C2H2O2S), were determined by Electrospray ionization (ESI) mass spectroscopy. These nanoclusters possess a 28% quantum yield, which is higher than those of green-emitting Au and Ag nanoclusters. We also found that Pt nanoclusters have considerably low cytotoxicity and biocompatibility, and demonstrated that they could be used for biomedical imaging. This study provides the possibility to extend the photoluminescent wavelength of Pt nanoclusters to the near infrared region, which is ideal for biological imaging applications.
©2013 Optical Society of America
1. Introduction
The development of luminescent nanomaterials as fluorescent labels is an attractive research field owing to the promising application in biomedical imaging, biosensing, drug delivery, and disease diagnostic. Noble-metal nanoclusters [1–41], composed of several atoms, have been investigated as a new class of bioimaging probes. In particular, several groups have dedicated their efforts in developing series of fluorescent Au [1–3,15] and Ag [3,23,24] nanoclusters, which have emission wavelengths ranging from UV to near infrared (NIR) [9] light, for biomedical imaging [3,15–19,33–35] and biosensing [3,11–14,29–32]. Recently, fluorescent Pt nanoclusters have been also developed by our group [36], Kawasaki et al. [37], and X. L. Guével et al. [38]. In our previous paper, we reported the synthesis of blue-emitting Pt5 nanoclusters that have an 18% absolute quantum yield in water (excitation: 380 nm, emission: 470 nm) [36]. These Pt5 nanoclusters were prepared by reducing Pt/PAMAM (G4-OH) solution with NaBH4 at room temperature. The products were then purified from PAMAM (G4-OH) and other chemical species by high performance liquid chromatography (HPLC), resulting in atomically monodispersed Pt5 nanoclusters. We also showed these nanoclusters could be used for cellular imaging and have low cytotoxicity. However, their excitation by UV irradiation risks autofluorescence, phototoxity and strong light scattering. Therefore, we sought nanoclusters with longer wavelength emissions for bioimaging purposes. In this paper, we describe a new scheme for synthesizing green-emitting Pt nanoclusters by reducing Pt ions gradually from pre-equilibrated Pt/PAMAM (G4-OH) complexes using a milder reductant (trisodium citrate) than NaBH4. Importantly, based on Pt/PAMAM (G4-OH) complex absorbance measurements before reduction, we found our pre-equilibration method could trap more Pt ions in PAMAM (G4-OH) and prepared larger nanoclusters than previous methods [36].
2. Synthetic method
PAMAM (G4-OH) (Sigma-Aldrich) (0.25 µmol) was added to 5 mL of millipore water (18.2 MΩ) and then mixed with H2PtCl6 (Wako Pure Chemical Industries (Japan)) (0.5 M, 30 µL). The Pt ions are coordinated with the interior tertiary amines of the PAMAM dendrimers [42,43]. The reaction mixture was allowed to stand in the dark at 4°C to minimize Pt ion oxidization of the PAMAM dendrimers [42,43]. Since the ligand-to-metal charge transfer (LMCT) UV absorption at 250 nm indicates the complexation of Pt ions with PAMAM (G4-OH), we measured UV–Vis absorbance spectrum of the Pt/PAMAM (G4-OH) solution during incubation by using an UV-2450 spectrophotometer (SHIMAZDU) and quartz cell with 1 cm path length. The background spectrum was subtracted by using an identical cell filled with millipore water (18.2 MΩ).
Figure 1(a) illustrates the time evolution of absorbance at 250 nm. Although the LMCT band at 250 nm sharply decreased in the first 3 hours, it slowly increased thereafter, reaching its maximum in the first day (24 h). After that, the absorbance gradually decreased again. On the other hand, as shown in Fig. 1(b), the absorbance of PtCl62- at 262 nm [44] and that of PAMAM (G4-OH) at around 200 nm precipitously decreased in the first 3 hours, and slowly changed thereafter. In the first 3 hours, Pt(IV) ions interacted with the external tertiary amines of PAMAM (G4-OH) and were reduced to Pt(II) ions. This reaction corresponds to decrease in absorbance at 262 nm and 200 nm. When Pt(II) ions formed coordination bonds with theinternal tertiary amines of PAMAM (G4-OH), the absorbance of the LMCT band at 250 nm increased. Complexation of Pt(II) ions with PAMAM (G4-OH) reached the equilibrium after 24 hours. We believe the coordination bonds are broken when Pt(II) ions oxidize PAMAM (G4-OH). This result indicates that the number of Pt ions complexed with PAMAM dendrimers reaches the maximum approximately one day after the reaction started. Therefore, we decided that the reduction reaction started 24 hours after pre-equilibrating the Pt-PAMAM complex. We added a reductant (trisodium citrate (Wako Pure Chemical Industries (Japan)); 1 M, 300 µL) to the pre-equilibrated Pt/PAMAM (G4-OH) solution, and incubated this reaction mixture at 90°C for two weeks under continuous stirring to form nanoclusters [45]. No precipitates were observed after incubation. Then, ultracentrifugation (Optima MAX-XP Benchtop Ultracentrifuge, Beckman Coulter, Inc.; 100,000 G) was performed for 30 min at 4°C to remove Pt colloidal nanoparticles.
Fig. 1 (a) Time evolution of absorbance for Pt/PAMAM (G4-OH) complexation at 250 nm. (b) UV-Vis spectra showing the complexation of Pt ions with PAMAM (G4-OH): the absorbance at 250 nm corresponds to the LMCT band. The left and right arrows show the decrease in the absorbance of PAMAM (G4-OH) at around 200 nm and that of PtCl62- at 262 nm, respectively.
3. Purification
After ultracentrifugation, mercaptoacetic acid (Wako Pure Chemical Industries (Japan)) (0.1 M, 3.0 µL) was added to the supernatant (300 µL) to replace PAMAM (G4-OH) (50 µM) with mercaptoacetic acid in the Pt nanocluster complex. The reaction mixture was allowed to stand at room temperature for 3 days. The PAMAM (G4-OH)/mercaptoacetic acid molar ratio was set to 1:20. The supernatant to which mercaptoacetic acid was added was separated into four fractions by using size-exclusion HPLC (Fig. 2(a) ). We used the HPLC system with the same condition as our previous work [36]. After collecting the fractions, we measured the excitation-emission matrices spectra with a spectrofluorophotometer (RF-5300PC, SHIMAZDU). The first broad fraction (fraction 1), eluted from 28 min to 38 min during the retention time, showed no fluorescence (data not shown). While the second fraction (fraction 2) had fluorescence at around 420 nm (Fig. 2(b)), the third fraction (fraction 3) emitted fluorescence at around 420 nm and 520 nm (Fig. 2(c)). The fourth fraction (fraction 4) exhibited a single fluorescent component at around 520 nm (Fig. 2(d)).
Fig. 2 (a) Size-exclusion HPLC chromatogram of the supernatant to which mercaptoacetic acid was added after centrifugation. HPLC was monitored by UV absorption at 290 nm (red line) and fluorescence at 520 nm (green line). Excitation-emission matrices spectra of fraction 2 (b), 3 (c) and 4 (d). (e) Excitation (blue line) and emission spectra (green line) of the Pt8 nanoclusters in water. (f) Fluorescent image of the Pt8 nanoclusters in water under UV (365 nm) irradiation.
4. Characterization
Furnace atomic absorption spectrometry (AA-6700F (SHIMAZDU)) was implemented with the 266 nm line for the fractions 2 to 4. We found only Pt to be present in fraction 3 (5.74 mg/L) and 4 (1.64 mg/L), while we did not detect Pt in fraction 2.
ESI mass spectrometry was then performed to determine the molecular weight of the chemical constituents in the fraction 4 by using LTQ XL (Thermo Fisher Scientific K.K.). The fraction 4 was dissolved in a 50% (v/v) water/methanol solution for measurement [28]. As shown in Fig. 3 , the main peak was detected at m/z = 2353.22, which is assigned to [Pt8L8 + 3Na + 4H]- (L = C2H2O2S). From the results, we found that the synthesized nanoclusters composed eight platinum atoms and that they were monodispersed. The fluorescent peaks (excitation: 460 nm, emission: 520 nm) observed in Fig. 2(c) and Fig. 2(d) originate from Pt nanoclusters, while the peaks (excitation: 330 nm, emission: 420 nm) observed in Fig. 2(b) and Fig. 2(c) are attributed to PAMAM (G4-OH), of which fluorescence was reported in [36] and [46]. We deduce from these results that fraction 4 contains Pt8L8 whereas fraction 3 contains Pt8 nanoclusters enclosed in PAMAM (G4-OH). Finally, Pt8 nanoclusters were found to have longer wavelength emissions than Pt5 nanoclusters [36].
Fig. 3 ESI mass spectrum of Pt nanoclusters. The peak, m/z = 2353.22, is assigned to [Pt8L8 + 3Na + 4H]- (L = C2H2O2S), and shows Pt nanoclusters consist of eight platinum atoms.
Then, we evaluated the fluorescent lifetime of these Pt nanoclusters using fluorescent lifetime measurement system. Femtosecond pulses from a regenerative amplifier (Libra HE, Coherent) were led to an optical parametric amplifier (OPerA Solo, Coherent), which generates 60 fs pulses at 1 kHz repetition rate. The beam was directed to two BBO crystals to generate a third harmonic pulse with a wavelength of 460 nm. The excitation light was softly focused onto samples placed in 1 cm cuvettes. The power density of the excitation beam was set to ~5 μJ/cm2, which is sufficiently low to avoid any saturation effects. Time-resolved spectra were obtained using a photon-counting streak camera (C4780, Hamamatsu Photonics, Japan) through a 25-cm monochromator (250is, Chromex). The measured fluorescence lifetime of green-fluorescence Pt nanoclusters was 6.5 ± 0.5 ns (Fig. 4 ), which was obtained by single exponential fitting. The fluorescence decay curve matches well to the single exponential decaying function, which indicates that the synthesized Pt nanoclusters have single molecular structure. Next, we evaluated the photobleaching characteristic of the Pt8 nanoclusters by observing decay in fluorescence intensity. The fluorescence intensity decay was measured and fitted by a single exponential function, F(t) = F0exp(-t/Td) (t: irradiation time, F0: fluorescence intensity at t = 0, Td: decay time which corresponds to F(Td)/F0 = 1/e). Decay time of Pt8 nanoclusters was 125 min, while that of organic fluorophore (1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyamine iodide (DiI)), which is well-known as a high resistant fluorophore to photobreaching, was 50 min. We found that the Pt8 nanoclusters were less subject to photobleaching than DiI. Furthermore, we examined the absolute quantum yield (QY) of the Pt nanoclusters with a QY measurement system (C10027, Hamamatsu Photonics, Japan). The Pt8 nanoclusters exhibited a QY of 28% in water, which betters the 18% QY of Pt5 [36]. This value also exceeds those of other green-emitting nanoclusters such as Au (QY = 25%, em = 510 nm) [1,2] and Ag (QY = 16%, em = 520 nm) nanoclusters [23]. We consider that the molecular structure of Pt nanoclusters and ligands affect their electronic properties, which might result in broadening of excitation/emission spectra and high QY. In order to elucidate their luminescence mechanism in detail, we will perform molecular orbital calculation in future.
Fig. 4 Fluorescence lifetime of Pt8 nanoclusters: the fluorescence lifetime of Pt8 nanoclusters was obtained by single exponential fitting (6.5 ± 0.5 ns).
5. Application to bioimaging
We investigated the feasibility of our green-emitting Pt nanoclusters as fluorescent probes for cellular imaging. In this study, we employed human epithelial carcinoma HeLa cells (DS Pharma Biomedical Co., Ltd.) in which chemokine receptors are highly expressed [47]. We conjugated Pt8L8 with an anti-chemokine receptor (anti-CXCR4-Ab) antibody (BioLegend, Inc.) by using Protein A (Thermo Fisher Scientific K.K.) as the adapter protein [36,48,49]. Conjugation protocol was described in our previous work [36]. HeLa cells were labeled with Pt nanoclusters in cell culture medium for 5 min prior to cell imaging. As we did not observe any change on their fluorescence properties, Pt nanoclusters exhibited stability in cell culture medium. Confocal fluorescent imaging was performed with FV1000 (Olympus) microscope using an oil immersion objective lens (40×, N.A. = 1.30), a 473 nm excitation laser and an appropriate emission filter (490-540 nm). Figure 5(a) shows a confocal fluorescent image of HeLa cells labeled with Pt8 nanoclusters. Green fluorescence (520 nm) was observed on the cellular membranes of HeLa cells treated with Pt8 nanoclusters. Conversely, no fluorescent signal was detected in a control sample labeled without the Pt8 nanoclusters (Fig. 5(b)). Therefore, the observed fluorescence in the presence of Pt8 nanoclusters is not autofluorescence from the HeLa cell. In addition, we investigated the cytotoxity of Pt8 nanoclusters in living HeLa cells by the same cell viability test as our previous work [36]. After HeLa cells were incubated with Pt8 nanoclusters (100 nM) for 48 h, we found that more than 97% of cells were alive. This value was comparable to that for the control sample without Pt8 nanoclusters. These results showed that Pt8 nanoclusters have very low cytotoxicity, and verify that Pt8 nanoclusters are relatively harmless fluorescent probes that can be used for the long-term imaging of living cells.
Fig. 5 Laser confocal fluorescent microscopic image overlaid with differential interference contrast images of living HeLa cells labeled with (a) and without (b) (Pt8L8)-(ProteinA)-(anti-CXCR4-Ab).
6. Conclusion and discussion
We synthesized green-emitting Pt8 nanoclusters (excitation: 460 nm, emission: 520 nm) that achieved a 28% QY. A key to this method is the pre-equilibration of the Pt ions with PAMAM dendrimers, which promotes the formation of coordination bonds between the Pt ions and the tertiary amines groups of the PAMAM dendrimers and the number of Pt ions trapped in the PAMAM dendrimers increases. Since the number of the tertiary amines groups are increased by using a higher generation of dendrimer, more Pt ions can be coordinated with the higher generation of PAMAM dendrimers. Therefore, synthesis of larger Pt nanoclusters opens the door to realize longer wavelength photoluminescence with such a higher generation of dendrimer. Protein such as bovine serum albmin (BSA) [6] and ferritins [19] can be also another candidate for a molecular template to prepare larger Pt nanoclusters, because they possess a nanometer-sized cavity and have already been utilized to synthesis red- and NIR-emitting Au nanoclusters. Using this method, we expect to extend the photoluminescent wavelength of Pt nanoclusters to NIR region, which is often preferable for in vivo imaging experiments. NIR-emission is especially suitable for whole body imaging application, disease diagnostics and clinical setting, where low background autofluorescence and deep penetration depth are desirable characteristics. We will also demonstrate the feasibility of Pt nanoclusters as a probe for high resolution microscopy such as two photon excitation microscopy and stimulated emission depletion (STED) microscopy in future.
Acknowledgments
This work is supported by the Osaka University Graduate School of Frontier Biosciences Global COE program. One of the authors, Y. Inouye, gratefully acknowledges financial support by a Grant-in-Aid for Scientific Research No. 24360026 from the Ministry of Education, Culture, Sports, Science and Technology. The authors thank Dr. P. Karagiannis for checking the manuscript, Dr. M. Murakami for ESI mass spectrometry, Dr. T. Fukumoto for furnace atomic absorption spectrophotometry, and Prof. K. Namba and Dr. T. Kato for allowing us to use their ultracentrifugation system.
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