Rare observation of ‘aggregation induced emission’ in cyclometalated platinum(II) complexes and their biological activities

Abstract

Three strong solid state emissive cyclometalated platinum(II) complexes [Pt(C⁁N) (CH⁁N) (Cl)] (1) (C⁁N/CH⁁N = 2-phenylpyridine, C⁁N = bidentate and CH⁁N = monodentate), [Pt(C⁁N) (P⁁P)]Cl [P⁁P = bis(diphenylphosphino)ethane (2) and cis-1,2-bis(diphenylphosphino)ethene (3)] were reported. These were identified as ‘Aggregation Induced Emission (AIE)’ active complexes based on controlled experiments. Cytotoxicity and cell imaging have been studied for the complex 2.

---

Phosphorescent heavy metal complexes like platinum(II) and iridium(III) are becoming increasingly important to scientists with respect to their applications in different fields such as, bioimaging,¹ sensing² and organic light emitting devices (OLEDs)³ due to their high luminescence quantum yields, color tunability, fair stability, excellent emission properties and straightforward synthetic routes.⁴ In these cases, 100% internal quantum efficiency can be achieved due to strong spin orbit coupling.⁵ The strong emission of the luminophores (organic as well as organometallic complexes) often quenched in their aggregate form called ‘Aggregation Caused Quenching (ACQ)’ effect.⁶ This is one of the major challenges to apply these materials in practical applications. In 2001 Tang and co workers⁷ achieved tremendous success in developing the anti-ACQ flourophores called ‘Aggregation Induced Emission (AIE)’ compounds. This emission phenomenon is manifested by compounds exhibiting significant enhancement of their light-emission in solid state whereas weak emission in solution. To date, many AIE flourophores have been reported⁸ but the development of heavy metal complexes with AIE properties is still limited.⁹ Out of the heavy metal complexes, reports of platinum(II) complex with this unusual property is rare.¹⁰ Yam and Che reported¹¹ AIE active Pt(II) complexes. The proposed mechanism of AIE in case of Pt(II) is restricted intramolecular rotation (RIR),¹² hydrogen bonding^10a which can lead to suppression in molecular motion. Apart from these, significant contribution of MMLCT excited state to the lowest excited states lead to strong emission in solid state.¹²

The normal luminophores suffer from basic problems like interference from background and scattered light.¹³ The development of AIE active Pt(II) complexes is effective solution to overcome these problems. The AIE active Pt(II) compounds with such properties as low photo-bleaching, low light scattering and rich photophysical and strong emission in the solid state make these promising candidates for bioimaging.^10b

Herein, we reported the syntheses of three AIE active cyclometalated complexes [Pt(C⁁N)(CH⁁N)Cl] (1), Pt(C⁁N)P⁁P]Cl where [C⁁N = 2-phenylpyridine; P⁁P is bis(diphenylephasphino)ethane (2) and cis-1,2-bis (diphenylephasphino)ethene (3), studied their photophyscial properties. The computational studies of one of the complexes was performed and correlated with its spectroscopic observations. All these complexes are found to exhibit AIE activity and emit very strongly in the solid state. Complex 1 is used for cytotoxicity study against non-resistant and cis-platin-resistant cell line showing very good results.¹⁴ This result encouraged us to go for MTT cytotoxicity study for the rest of the synthesised complexes. The MTT cytotoxicity study has been carried out for complex 2. Utilizing its AIE and rich photophysical properties, we have used complex 2 in bio-imaging applications as in staining ability of cancer cells, human hepatocellular carcinoma cells, Hep3B.

The syntheses of all three complexes 1, 2 and 3 are presented in Scheme 1. The green synthetic approach has been applied for the syntheses of these complexes. Complex 1 has been synthesized by using K₂PtCl₄ as the platinum precursor along with four equivalents of 2-phenyl pyridine. The reaction was completed using water as the solvent in presence of microwave (MW) in 10 minutes. The greenish yellow product, 1 was isolated from water as a solid mass and characterized by ¹H and ¹³C NMR (Fig. S1 and S2†). The synthesized pendent complex 1 is one of the important precursors for synthesis of many luminescent Pt(II) complexes.¹⁵

Scheme 1 Synthetic route and chemical structures of complexes 1, 2 and 3.

The synthesis of complex 2 was reported¹⁶ by M. G. Haghighi et al. using two step synthetic protocol with using of unusual platinum(II) precursors, (i) [PtMe(κ¹C-ppy)(dppe)] (ii) [Pt(ppy)(CF₃CO₂)(SMe₂)]. Herein, the syntheses of bis-chelate phosphine complexes 2 and 3 were carried out using complex 1 in a facile and in very short reaction time. Reaction between 1 and bis(diphenylphosphine)ethene (dppe)/bis (diphenylphosphine)ethylen (dppen) in (1 : 1) ratio resulted complexes 2 and 3 after stirring the reaction mixture for 1 minute at room temperature in dichloromethane (DCM). The complexes 2 and 3 were characterized by ¹H, ¹³C and ³¹P NMR.

¹H NMR spectra of complex 2 shows aromatic proton signals in the range δ = 6.8–8.3 ppm, the four protons of (CH₂–CH₂) of the dppe ligand were observed as multiplets at δ = 2.63 ppm (Fig. S3†). The ¹³C NMR spectra of the complexes 2 and 3 correspond to their structure (Fig. S4 and S5†). The ³¹P NMR of this complex shows two distinct singlets at δ = 41.39, for the P trans to N with ¹J(_PtP) = 3772 Hz, and δ = 51.16, for the P trans to C with a much lower value of ¹J(_PtP) = 1877 Hz due to the trans influence of C being much greater than that of N. The range of ¹J_(PtP) between 1877–3762 Hz indicates cis coordination of the phosphine ligand¹⁷ (Fig. S6†). The ethylene (CH=CH) proton signal for the dppen ligand appears at δ = 7.2 ppm as a multiplate¹⁸ (Fig. S7†). The ³¹P spectrum of complex 3 shows two doublet of doublets, one at δ = 43.8 ppm with ³J_{(PP b)} = 17 Hz and ¹J_{(PtP a)} = 3782 Hz for P trans to N, and δ = 59.0 ppm with ³J_{(PP a)} = 17 Hz and ¹J_{(PtP b)} = 1839 Hz for P trans to C (Fig. 1), the lower value of ¹J_(PtP) suggests trans effect of carbon with respect to nitrogen.

Fig. 1 ³¹P NMR spectrum of [Pt(Pppy) (dppen)]Cl (3) in CDCl₃.

The complexes are soluble in solvents such as, dichloromethane (DCM), dimethyl formamide (DMF), 1,4 dioxane, methanol, acetonitrile etc, but are insoluble in water as well as in hexanes. These complexes show very week emission in all these solvents but they are intensely emissive in the solid state. These observations hint that all three complexes are expected to show AIE behaviour. In order to investigate this AIE property, water was used as poor solvent for complex 1 and hexane for complexes 2 and 3.

Different amounts of water fraction (f_w = 0–90%) were added to the pure THF solution of 1 with keeping the same concentration of each solution to 1 × 10⁻⁵ M. The emission intensity increased gradually with increasing f_w resulting in a cloudy solution indicating the formation of aggregates. The maximum emission intensity was observed with f_w = 90% which was 12.5 times higher than the intensity of its solution in pure THF (Fig. S8†).

For complexes 2 and 3, different amounts of hexane fraction (f_h = 0–90%) [Fig. 2i–vi] were added to their solutions, (keeping the concentration of each solution remains same to 5 × 10⁻⁵ M). Maximum emission intensity was observed at f_h = 90%, for both the complexes. The emission intensity was increased by 19 times for 2 and 20 times for 3 as compared with their original respective solution intensities. The solid vs. solution PL emission spectra (in THF) for the complexes 1, 2 and 3 are shown in Fig. S9–S11.† The solid state absolute quantum efficiency (QE) for the complexes 2 and 3 were measured and found to 0.253 and 0.247, respectively. The solution quantum efficiency were found to be 0.001 and 0.0009, respectivley (Table S1†). So, the solid state QE for the complexes 2 and 3 rises to ϕ_solid/ϕ_solution > 250 (i.e., 2 and 3 show 253 and 274 times higher QE than their respective solutions states). There was very significant rise of QE observed as far as the AIE property of the complexes are concerned.

Fig. 2 PL spectra of 2 and 3 in DCM/hexane mixed solvents with different f_h with excitation at 385 nm [for (i) and (iv) for 2 and 3, respectively], (λ_max: a = 479 and b = 512 nm for 1 and a = 490 and b = 517 nm for 2).

To understand the origin of the AIE property, the crystal-packing of 1 and 2 were examined (Fig. 3 and S12;† Table S2 and S3†). The crystal structure of complex 1 shows short contacts, which are mainly C–H⋯π type of interactions and falls in the range of 2.64–2.85 Å (ref. 15d) (Fig. 3a). This is shorter than the sum of van der Waals radius of C and H. The crystal structure of 2 exhibits many C–H⋯π interactions in the range of 2.81–2.99 Å (Fig. 3b). These interactions may be responsible for restricted rotation of the phenyl rotors present in these molecules in their solid states and hence the complexes exhibit AIE activity.

Fig. 3 (a) Crystal packing diagram of complex 1 showing C–H⋯π type short contacts, H2–Cg1 = 2.85 Å and H8–Cg2 2.64 Å (b) crystal packing diagram of complex 2 short contacts H1B–Cg5 = 2.81, H24–Cg8 = 2.93, H28–Cg3 = 2.92, H29–Cg2 = 2.99 and H9–Cg5 = 2.96 (the counter ion is omitted for clarity).

Variation of PL intensity with respect to changes of wavelength with inclusion of error bar [(ii) and (v) for 2 and 3, respectively]; luminescent images of 2 and 3 [(iii) and (vi) for 2 and 3, respectively] (irradiated with an ultraviolet light at 365 nm) in hexane–DCM mixed solvents with the concentration kept at 2 × 10⁻⁵ mol L⁻¹.

The computed energy gap between ground singlet state and first excited singlet state is 362.6 nm (Fig. 4). This is in agreement with experimental absorption wavelength from spectroscopic study.

Fig. 4 UV-Visible absorption and photoluminescence spectra of complex 2 in 1 × 10⁻⁵ M DCM.

The oscillator strength is high as compared to previously reported¹⁹ iridium(III) complexes indicating stronger singlet to singlet absorption. The energy gap between ground singlet state to first excited triplet state (491.1 nm) (Fig. 4) is in accordance with emission wavelength obtained from spectroscopy. Although emission is an excited state property, results based on ground state optimization qualitatively describes the process.²⁰ Assignments of transitions (Table S4†) show the relative involvement of different frontier orbitals in absorption and emission spectra. Natural Bond Orbital (NBO) analyses have revealed that chlorine atom makes major contribution towards HOMO, along with platinum(II). Whereas LUMO is exclusively distributed over the fused ring attached to platinum(II) (Fig. 5).

Fig. 5 Frontier orbitals for Platinum complex. Major contribution of HOMO comes from the counter ion, chlorine. LUMO is distributed over the fused ring. These orbitals are obtained from DFT calculations of the Platinum complex after ground state optimization. Calculations are performed by GAMESS US software. Visualization of the molecular orbital is through MOLDEN software.

This data along with the assignments, indicate the strong possibility of metal to ligand charge transition (MLCT) in this complex (Table S4†).

Biological applications: human hepatocellular carcinoma cells, Hep3B were treated with increasing doses of platinum compound, 2 for varied time points and the cell viability was determined through several assays. As shown in Fig. 6, the platinum compound showed increased cytotoxicity in Hep3B cells in a dose-dependent manner as determined by MTT assay. The IC₅₀ of the compound was found to be around 5 μM at 24 h. Similar comparable results were obtained with WST-1 (Fig. S13†) and Trypan Blue assay (data not shown) performed to validate results from MTT assay. Time kinetic study was also performed by incubating the cells with increasing doses of platinum compound for 24, 48 and 72 h. With increase in time of treatment with platinum compound, the cell viability was found to be significantly decreased (Fig. 6).

Fig. 6 The time kinetic study of complex 2 as assayed by MTT.

Further, this compound was successfully checked for cellular internalization potential through fluorescence imaging of live Hep3B cells (Fig. 7). An exclusive staining of the nucleus of live cells was observed; the platinum compounds are well known for their ability to form DNA-adducts. We speculate that the cytotoxicity that we observed in Hep3B cells can be attributed to the increased internalization potential of the drug and also to its property of binding to cellular DNA. Hence, the use of this compound provides one with dual option, not only for its use as an anti-cancer drug, but also as a cell-visualization or bio-imaging agent because of its fluorescence upon aggregation property and good cell membrane permeability.

Fig. 7 Bright field image (a) and fluorescence image (b) (right, 100×) of Hep3B cells following treatment with complex 2. Fluorescence image depicts successful internalization of the complex by Hep3B cells and its AIE characteristics [Scale bar (5 μm)].

A facile and greener synthetic methodology has been developed for the synthesis of these AIE active cyclo-metalated platinum(II) complexes. The synthesized complexes are also showing ‘aggregation induced emission (AIE)’ behaviour. In our laboratory, we are currently exploring the possible options to modify this compound for enhanced targeting of specifically cancer cells in vivo, compared to normal. Furthermore, we are also channelizing our future research to understand the molecular mechanism of anti-cancer activity of the compound so that they can be tagged with appropriate adjuvant to increase specificity and anti-cancer activity.

Acknowledgements

We thank the ‘Department of Science and Technology (DST), Govt. of India’ for financial support under two projects (no: SR/S1/IC-48/2009 and SB/S1/IC-13/2014) and Council of Scientific and Industrial Research (CSIR) no. 01/2551/12/EMR-II. We also acknowledge ‘UGC-sponsored Special Assistance Programme (F.540/14/DRS/2007, SAP-I)’ and DST FIST for the instrumental support. We thank Nigam P. Rath, Department of Chemistry & Biochemistry and Centre for Nanoscience, University of Missouri-St. Louis for providing single crystal X-ray diffraction facility. We thank IISER Mohali, India for prividing NMR facility. Special thank is given to Dr Ashish Gupta, Samtel Centre for Display Technologies, IIT Kanpur, India for providing the facility of solid state quantum yield measurement.

Notes and references

1. (a) Q. Zhao, T. Y. Cao, F. Y. Li, X. H. Li, H. Jing, T. Yi and C. H. Huang, Organometallics, 2007, 26, 2077; (b) Q. Zhao, F. Y. Li and C. H. Huang, Chem. Soc. Rev., 2010, 39, 3007; (c) X. Mou, Y. Q. Wu, S. J. Liu, M. Shi, X. M. Liu, C. M. Wang, S. Sun, Q. Zhao, X. H. Zhou and W. Huang, J. Mater. Chem., 2011, 21, 13951.
2. (a) Q. Zhao, F. Li and C. Huang, Chem. Soc. Rev., 2010, 39, 3007; (b) M. L. Ho, Y. M. Cheng, L. C. Wu, P. T. Chou, G. H. Lee, F. C. Hsu and Y. Chi, Polyhedron, 2007, 26, 4886; (c) Q. Zhao, S. J. Liu, F. Y. Li, T. Yi and C. H. Huang, Dalton Trans., 2008, 3836; (d) G. G. Shan, H. B. Li, H. Z. Sun, D. X. Zhu, H. T. Cao and Z. M. Su, J. Mater. Chem. C, 2013, 1, 1440.
3. (a) J. D. Slinker, A. A. Gorodetsky, M. S. Lowry, J. Wang, S. Parker, R. Rohl, S. Bernhard and G. G. Malliaras, J. Am. Chem. Soc., 2004, 126, 2763; (b) P. T. Chou and Y. Chi, Chem.–Eur. J., 2007, 13, 380; (c) B. Ma, P. I. Djurovich and M. E. Thompson, Coord. Chem. Rev., 2005, 249, 1501; (d) S. R. Forrest and M. E. Thompson, Chem. Rev., 2007, 107, 923.
4. W.-Y. Wong and C.-L. Ho, Coord. Chem. Rev., 2009, 253, 1709.
5. (a) B. Tong, Q. Mei, S. Wang, Y. Fang, Y. Menga and B. Wang, J. Mater. Chem., 2008, 18, 1636; (b) M. A. Baldo, D. F. O'Brien, Y. You, A. Shoustikov, S. Sibley, M. E. Thompson and S. R. Forrest, Nature, 1998, 395, 151; (c) M. A. Baldo, S. Lamansky, P. E. Burrows, M. E. Thompson and S. R. Forrest, Appl. Phys. Lett., 1999, 75, 4; (d) E. Turner, N. Bakken and J. Li, Inorg. Chem., 2013, 52, 7344.
6. C. H. Huang, F. Y. Li and W. Huang, Introduction to Organic Light-Emitting Materials and Devices, Press of Fudan University, Shanghai, 2005.
7. Y. Hong, J. W. Y. Lam and B. Z. Tang, Chem. Soc. Rev., 2011, 40, 5361.
8. (a) T. Han, X. Feng, B. Tong, J. Shi, L. Chen, J. Zhi and Y. Dong, Chem. Commun., 2012, 48, 416; (b) X. T. Chen, Y. Xiang, N. Li, P. S. Song and A. j. Tong, Analyst, 2010, 135, 1098.
9. (a) Q. Zhao, L. Li, F. Y. Li, M. X. Yu, Z. P. Liu, T. Yi and C. H. Huang, Chem. Commun., 2008, 685; (b) P. Alam, M. Karanam, A. Roy Choudhury and I. R. Laskar, Dalton Trans., 2012, 41, 9276; (c) K. W. Huang, H. Z. Wu, M. Shi, F. Y. Li, T. Yi and C. H. Huang, Chem. Commun., 2009, 1243; (d) C. H. Shin, J. O. Huh, M. H. Lee and Y. Do, Dalton Trans., 2009, 6476.
10. (a) S. Liu, H. Sun, Y. Ma, S. Ye, X. Liu, X. Zhou, X. Mou, L. Wang, Q. Zhao and W. Huang, J. Mater. Chem., 2012, 22, 22167; (b) H. Honda, Y. Ogawa, J. Kuwabara and T. Kanbara, Eur. J. Inorg. Chem., 2014, 1865.
11. (a) V. W.-W. Yam, K. M.-C. Wong and N. Zhu, J. Am. Chem. Soc., 2002, 124, 6506; (b) W. Lu, Y. Chen, V. A. L. Roy, S. S.-Y. Chui and C.-M. Che, Angew. Chem., Int. Ed., 2009, 48, 7621.
12. M. X. Zhu, W. Lu, N. Y. Zhu and C. M. Che, Chem.–Eur. J., 2008, 14, 9736.
13. (a) H. Yoshika and K. Nakatsu, Chem. Phys. Lett., 1971, 11, 255; (b) B. K. An, D. S. Lee, J. S. Lee, Y. S. Park, H. S. Song and S. Y. Park, J. Am. Chem. Soc., 2004, 126, 10232.
14. (a) T. Okada, I. M. El-Mehasseb, M. Kodaka, T. Tomohiro, K.-i. Okamoto and H. Okuno, J. Med. Chem., 2001, 44, 4661; (b) I. M. El-Mehasseb, M. Kodaka, T. Okada, T. Tomohiro, K.-i. Okamoto and H. Okuno, J. Inorg. Biochem., 2001, 84, 157.
15. (a) M. M. Mdleleni, J. S. Bridgewater, R. J. Watts and P. C. Ford, Inorg. Chem., 1995, 34, 2334; (b) J.-Y. Cho, K. Y. Suponitsky, J. Li, T. V. Timofeeva, S. Barlow and S. R. Marder, J. Organomet. Chem., 2005, 690, 4090; (c) D. M. Jenkins and S. Bernhard, Inorg. Chem., 2010, 49, 11297; (d) N. Godbert, T. Pugliese, I. Aiello, A. Bellusci, A. Crispini and M. Ghedini, Eur. J. Inorg. Chem., 2007, 5105.
16. M. G. Haghighi, S. M. Nabavizadeh, M. Rashidi and M. Kubicki, Dalton Trans., 2013, 13369.
17. (a) F. Raoof, A. R. Esmaeilbeig, S. M. Nabavizadeh, F. N. Hosseini and M. Kubicki, Organometallics, 2013, 32, 3850; (b) S. M. Nabavizadeh, H. R. Shahsavari, M. Namdar and M. Rashidi, J. Organ. Chem., 2011, 696, 3564; (c) M. G. Haghighi, M. Rashidi, S. M. Nabavizadeh, S. Jamali and R. J. Puddephatt, Dalton Trans., 2010, 11396.
18. R. H. Vaz, R. M. Silva, J. H. Reibenspies and O. A. Serra, J. Braz. Chem. Soc., 2002, 13, 82.
19. P. Alam, I. R. Laskar, C. Climent, D. Casanova, P. Alemany, M. Karanam, A. R. Choudhury and J. R. Butcherd, Polyhedron, 2013, 53, 286.
20. P. Alam, M. Karanam, D. Bandyopadhyay, A. R. Choudhury and I. R. Laskar, Eur. J. Inorg. Chem., 2014, 3710.

---

Footnote

† Electronic supplementary information (ESI) available. CCDC 1007679. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4ra06623e

---