One-step rapid synthesis of fluorescent platinum nanoclusters for cellular imaging and photothermal treatment

Abstract

Fluorescent platinum nanoclusters constructed through one-step synthesis from chloroplatinic acid cross swiftly across carcinoma cell membranes for bio-imaging and photothermal treatment.

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Metal nanoparticles have attracted much interest in the past two decades in the fields of catalysis,¹ sensing,² photonics,³ and biolabeling, due to their unusual properties and potential applications.^4–6 More recently, the metallic nanoclusters (NCs) including gold, silver NCs were found to exhibit unique fluorescence properties and have been explored for bio-imaging and diagnosis.^7–16 Platinum nanoclusters (Pt NCs), as one of the most important noble metal NCs, were observed to generate strand breaks in DNA¹⁷ and on the other hand, its insoluble forms possess notable antioxidative capacity and the potentially low cytotoxicity,^18–21 suggesting its potential for promising bio-applications.

The traditional synthesis of Pt NCs usually utilize either electrochemical process (which is difficult to get uniform and stable solution) or sodium borohydride as the reducing agent, which may lead to the reaction difficult to control and hardly get homogeneous quantum dots due to the strong reducibility of sodium borohydride.^21,22 Tanaka et al. reported the synthesis of fluorescent platinum nanoclusters via the reduction of NaBH₄ and tried to utilize them for bioimaging.²¹ However, according to the report of this literature, to complete the synthesis, the relevant reaction had to take a long time and last for about two weeks to get the product, while Pt NCs obtained was not homogeneous. Hui-Fen Wu reported the preparation of platinum nanoparticles for the photothermal treatment of Neuro 2A cancer cells,²³ but the strong cytotoxicity and large particle size of PVP-capped Pt NCs limited their bio-applications. In view of these observations, in this study we have explored the possibility to utilize a new green one-step synthesis method to rapidly get platinum nanoclusters for some disease bioimaging and treatment. This green one-step synthesis of Pt NCs could be readily realized through collaborative reduction of ascorbic acid and glutathione with chloroplatinic acid as precursor, where uniform Pt NCs can be obtained via rapid etching of GSH for the strong sulfydryl complexing ability. This simple and rapid one-step synthesis of Pt NCs can generate homogeneous and stable fluorescent nanoclusters. The as-prepared Pt NCs could be kept under 4 °C for six months without any change of the Pt NCs fluorescence, indicating its stability and promising application as efficient biomarker in future clinic diagnosis and treatment.

It is well known that cancer is still one of the most difficult diseases for the treatment, and photothermal cancer therapy based on nanomaterials has been paid much attention during recent years.^23,24 Nevertheless, the biocompatibility of the relevant nanomaterials is a demanding challenge during photothermal treatment. In this contribution we have explored a new strategy for the preparation and utilization of biocompatible fluorescent Pt NCs for the bio-labeling and photothermal treatment of target cancers. To the best of our knowledge, by now there have no report about the synchronization of bio-imaging and treatment of cancer cells by using Pt NCs.

As shown in Scheme 1, initially the Pt NCs was obtained through one-step synthesis from chloroplatinic acid under 80 °C for ca. 0.5 h or under 37 °C for a relatively longer time (48 h). The synthesized Pt NCs emitted a strong blue fluorescence under UV light (365 nm) irradiation. Thus, UV-vis absorption spectroscopy, fluorescence and FTIR spectroscopy could be readily utilized for monitoring the relevant synthesis process, as illustrated in Fig. 1a and Fig. S1.† It is observed that in comparison with that of the precursor chloroplatinic acid, UV-vis absorption spectra of the synthesized Pt NCs show the disappearance of absorption peak of 260 nm, while the fluorescence emission peak wavelength of the as-prepared Pt NCs was observed at 456 nm, which is well consistent with that reported previously in the literature by Tanaka²¹ and Kawasaki.²⁵ In addition, it is noted that the presence of GSH could efficiently facilitate the formation of Pt NCs and the relevant concentration of added GSH plays an important role for this synthesis of the nanoclusters (Fig. S1†). Evidence for the capped GSH on Pt NCs could be found from the FTIR spectroscopic study (Fig. S2†), where the relevant bands of GSH capped Pt NCs at ca. 3444 cm⁻¹, 2358 cm⁻¹ and 1647 cm⁻¹ could be attributed to the vibration stretching of relevant amino functional groups (i.e., –NH₂), sulfydryl and carboxylate anion –COO⁻, respectively.

Scheme 1 Schematic illustration of one-step rapid synthesis of fluorescent Pt NCs for tumor cell imaging and photothermal treatment.

Fig. 1 (a) Optical microscopy characterization of Pt NCs through the synergistic effect between ascorbic acid (VC) and glutathione (GSH) with chloroplatinic acid as precursor. (black solid line: UV-vis absorption spectrum of the relevant Pt NCs; blue solid line: fluorescence emission spectrum (Em) at a 390 nm excitation wavelength; red solid line: excitation spectrum.) (b) Typical images of TEM and HRTEM (inserted image) of Pt NCs, which is obtained by using the 1 : 1 mole concentration ratio of GSH to chloroplatinic acid, showing a lattice spacing of 3.92 ± 0.02 Å. (c) Hydrodynamic diameter measured using DLS (mean value ca. 1.4 nm).

A typical TEM image of the synthesized Pt NCs is shown in Fig. 1(b and c), evidencing their high monodispersity and uniform sizes of 1.4 ± 0.12 nm. TEM also established that the synthesized Pt NCs involved platinum atoms planes with an interplane distance of 3.92 ± 0.02 Å, which were almost spherical and had no noticeable trend to agglomerate. Energy dispersive spectroscopy (EDS) result showed that there is no other elemental impurity present in the nanoclusters (Fig. 2a). To further confirm the formation and valence state of Pt NCs, X-ray photoelectron spectroscopy (XPS) was implemented after drying the solution on a silicon wafer. As shown in Fig. 2, two peaks located at the binding energies of 75.1 eV and 71.8 eV were observed, which could be attributed to Pt 4f_5/2 and Pt 4f_7/2 peak, indicating acquisition of the reduced platinum Pt (0) (Fig. 2b). The binding energy of S 2p_3/2 located at 162.6 eV could be assigned to the binding of the sulfur atom to the platinum and Pt–S charge transfer in the thiol-coated platinum clusters²² (Fig. S3†).

Fig. 2 (a) EDS spectroscopy of Pt NCs. (b) X-ray photoelectron spectra (XPS) evidencing the Pt 4f photo-electron emission from platinum nanoclusters: one peak at 75.1 eV (Pt 4f_5/2) and the other at 71.8 eV (Pt 4f_7/2).

The mass spectra may provide some extra component information about additional details for the tentative structure of the NCs. For example, determined by ESI mass spectrometry upon dissolving the sample in a 50% (v/v) water system, it is evident that Pt NCs is mainly comprised of Pt cores and GSH ligands. The ESI data showed the high abundant components of [Pt₃(GS)₇]⁴⁻ at m/z = 682.17 (Fig. S4†). The high abundance for [Pt₃(GS)₇]⁴⁻ among all peaks indicates that Pt and GSH are prone to form complexes at a ratio of 3 : 7.

Based on the above observations, the bio-labeling and cytotoxic effect of Pt NCs on cancer cells was further explored, where HepG2 and HeLa cancer cells were chosen as relevant experimental models while non-cancerous ones L02 cells were chosen as the control model. As shown in Fig. 3 and Fig. S5,† when incubated with the Pt NCs for 24 h, the intracellular fluorescence of target cancer cells appears and many bright blue spots could be observed inside the cells. In contrast, when non-cancerous ones (L02 model) incubated with the Pt NCs, no apparent intracellular fluorescence could be observed. The above studies performed on cell cultures have evidenced that incubation of cancerous cells (HepG2 and HeLa models) and non-cancerous ones (L02 model) with Pt NCs led to drastic differences in relevant cell interactions. This could be attributed to the high affinity of GSH capped Pt NCs to cancer cells, where some specific biochemical characteristics of cancer cells differentiate them from cells under normal homeostasis. The intracellular fluorescence indicates that the Pt NCs could readily enter inside the target cancer cells, indicating the possibility for the promising use of these fluorescent Pt NCs for the direct recognition or bio-marking of cancer cells and potential monitoring of the relevant treatment process.

Fig. 3 Typical confocal laser scanning microscope images of the intracellular accumulation of Pt NCs in HepG2 cells. HepG2 cells were incubated in the absence ((a); control) or in the presence of Pt NCs 150 μg mL⁻¹ for 24 h (b), L02 cells were incubated in the presence of Pt NCs 150 μg mL⁻¹ for 24 h (c). Relative fluorescence intensity variations (d) along cross-sections A (in (a)), B (in (b)), or C (in (c)) (the color gradient coding illustrates the direction of the sampling) (scale bar are 25 μm).

Meanwhile, MTT assay for the cytotoxicity demonstrates that the Pt NCs possess very good biocompatibility and have little cytotoxicity under UV irradiation (Fig. 4a). However, the relevant Pt NCs possess strong lethality to cancer cells when using infrared (IR) irradiation (Fig. 4a(C)) under identical experimental conditions. Infrared irradiation could have much more efficient effect than that of UV irradiation during the Pt NCs induced photothermal treatment, indicating that it mainly depends on heating effect rather than free radical effect. Importantly, these Pt NCs show almost non-cytotoxic property to normal cells (i.e., L02 cells, Fig. 4a), suggesting it could be used to target cancer cells. Additionally, the morphology study by inverted fluorescence microscope illustrates the efficacy of the photothermal treatment of target cancer cells (Fig. 4 and S6†). As shown in Fig. S6,† the control experiments (i.e., with the Pt NCs treatment but without laser irradiation) or devoid of Pt NCs treatment as well as laser irradiation showed very marginal changes in the amount of adherent cells. In contrast, the relative laser treatment by using infrared (IR) irradiation in the presence of Pt NCs showed excellent HeLa cell-killing efficiency, accompanying with the distinct morphological changes and the reduced amount of adherent cells. In addition, tryphan blue staining were employed to confirm the killing efficacy of Pt NCs, and the cells with Pt NCs treatment as well as IR laser irradiation showed the present of cells stained blue (Fig. 4d), indicating the relevant good photothermal efficacy. By contrast, the cells are almost transparent in control groups, signifying that these cells were live and just laser irradiation or Pt NCs alone can hardly kill cells.

Fig. 4 (a) MTT assay of cytotoxic effect of Pt NCs on HeLa cells. (A) HeLa cells were treated with Pt NCs alone, (B) HeLa cells were treated with Pt NCs under UV irradiation (405 nm), (C) HeLa cells and (D) control (L02 cells) were treated with Pt NCs together with IR irradiation (635 nm) after incubation for 24 h at various concentrations of Pt NCs (i.e., 10, 20, 40, 60, 80, 160 and 320 μg mL⁻¹, etc.). (b) Effect of IR irradiation treatment on the viability of HeLa cells in the presence or without (control) Pt NCs (320 μg mL⁻¹) determined by MTT assay. (c) Tryphan blue staining of HeLa cells with laser treatment of IR irradiation for 10 min but without Pt NCs. (d) Tryphan blue staining of HeLa cells treated with Pt NCs (150 μg/10⁶ cells) and laser treatment of IR irradiation for 10 min (scale bar are 10 μm).

Conclusions

In summary, in this contribution the one-step rapid synthesis of fluorescent platinum nanoclusters has been realized through the synergistic effect between ascorbic acid (VC) and glutathione (GSH) with chloroplatinic acid as precursor. Based on the results from a variety of techniques, namely the MTT assay, fluorescence, FTIR spectroscopy, confocal laser scanning microscope as well as UV-vis absorption spectroscopy, it is evident that the green chemically synthesized Pt NCs could be readily utilized for the effective bio-imaging of cancer cells (i.e., HeLa and HepG2 cells) and for simultaneously efficient treatment of cancers through effect of IR irradiation. The Pt NCs based photo-theranostics owns good stability, high water dispersibility and solubility, non-cytotoxicity, and good biocompatibility, which may offer the possibility of the future utilization of Pt NCs in fluorescence imaging-guided photothermal therapy to realize the accurate bio-labeling and multimode treatments of target cancers.

Acknowledgements

This work is supported by the National Basic Research Program (2010CB732404) and National Natural Science Foundation of China (81325011, 21327902, 21175020), the National High Technology Research and Development Program of China (2012AA022703) and Suzhou Science & Technology Major Project (ZXY2012028).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: The experimental details and characterizations are included. See DOI: 10.1039/c4ra07121b

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