Multistimuli-Responsive Fluorescent Organometallic Assemblies Based on Mesoionic Carbene-Decorated Tetraphenylethene Ligands and Their Applications in Cell Imaging

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Feb 2022Multistimuli-Responsive Fluorescent Organometallic Assemblies Based on Mesoionic Carbene-Decorated Tetraphenylethene Ligands and Their Applications in Cell Imaging Yang Li, Tianfeng Yang, Nan Li, Sha Bai, Xin Li, Li-Li Ma, Kai Wang, Yanmin Zhang and Ying-Feng Han Yang Li Key Laboratory of Synthetic and Natural Functional Molecule of the Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an 710127 Google Scholar More articles by this author , Tianfeng Yang School of Pharmacy, Health Science Center, Xi’an Jiaotong University, Xi’an 710061 Google Scholar More articles by this author , Nan Li State Key Laboratory of Superhard Materials, College of Physics, Jilin University, Changchun 130012 Google Scholar More articles by this author , Sha Bai Key Laboratory of Synthetic and Natural Functional Molecule of the Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an 710127 Google Scholar More articles by this author , Xin Li Key Laboratory of Synthetic and Natural Functional Molecule of the Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an 710127 Google Scholar More articles by this author , Li-Li Ma Key Laboratory of Synthetic and Natural Functional Molecule of the Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an 710127 Google Scholar More articles by this author , Kai Wang State Key Laboratory of Superhard Materials, College of Physics, Jilin University, Changchun 130012 Google Scholar More articles by this author , Yanmin Zhang School of Pharmacy, Health Science Center, Xi’an Jiaotong University, Xi’an 710061 Google Scholar More articles by this author and Ying-Feng Han *Corresponding author: E-mail Address: [email protected] Key Laboratory of Synthetic and Natural Functional Molecule of the Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an 710127 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.021.202100780 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail The controllable construction of light-emitting organometallic supramolecular materials integrating stimulus responses and biological applications is still a major challenge today. In this work, emissive organometallic assemblies based on AgI/AuI and mesoionic carbene-decorated tetraphenylethene (MIC-TPE) ligands have been synthesized and characterized. Photophysical studies showed that these organometallic assemblies [M4( 2)2](OTf)4 not only fluoresce in various dilute solutions, resulting from the rigidification of MIC-TPE ligands upon complexation, but also show tunable fluorescence wavelengths and intensity behaviors in mixed solvent systems and at varying temperatures while in solution, and mechanical pressure stimulus-responsive fluorescent features in the solid state. Furthermore, the organometallic cages [M4( 2)2](OTf)4 (M = Ag and Au) were successfully employed for cell imaging and showed the potential of anticancer activity, making them promising candidates for cancer theranostics. This work provides a simple and efficient method to prepare highly emissive and stimulus-responsive organometallic materials and paves the way for related biological applications such as cancer cell imaging and targeted therapy. Download figure Download PowerPoint Introduction Stimuli-responsive luminescence supramolecular materials, materials that respond to light, pressure, temperature, or pH value, are of great interest owing to their appealing architectures1–3 and widespread applications across many different fields.4–12 The controllable fabrication of such materials and structures with high photostability, facile tunability, and good solubility in common organic solvents remains one of the current challenges in exploring the aforementioned applications.13,14 Traditional fluorophores usually suffer from aggregation-caused quenching (ACQ) in the solid state or form colloidal aggregates and exhibit the heavy atom effect when coordinated with transition metals.15–17 Fluorophores with aggregation-induced emission (AIE) properties have become excellent candidates for the construction of luminescent supramolecular materials.18 The tetraphenylethene (TPE) unit is a representative AIE building block that has great utility for the synthesis of different and discrete luminescent supramolecular structures and materials because of its facile syntheses and modification.19–22 Currently, a variety of luminescent supramolecular coordination complexes (SCCs), including metallacycles and metallacages containing TPE units, have been successfully developed from coordination-driven self-assembly.23–32 However, a highly emissive performance was only observed in their aggregate state because of effectively restricted rotation of the benzene rings, which is a typical AIE characteristic.33 The weak fluorescence performance of such materials in dilute solutions somewhat limits its applications. In parallel to the rapid development of SCCs constructed from metal acceptors and Werner-type organic donors,34–37 organometallic supramolecular assemblies, especially those constructed from poly-NHC (NHC = N-heterocyclic carbene) ligands and coinage metals (AgI and AuI), feature metal–carbon (M–CNHC) bonds and exhibit considerable advantages in the construction of luminescent organometallic supramolecular materials, which benefit from controllable structures and simple synthetic steps.38–41 Despite several recent reports of organometallic supramolecular complexes with high luminescence properties in solution and the solid state, the design and preparation of stimuli-responsive luminescent organometallic supramolecular materials, and their practical application, have not yet been exploited mainly because of their limited stability.41–43 Mesoionic carbene (MIC) ligands are a subclass of the NHC-family of ligands that have received widespread attention in recent years. In general, they have stronger electron donor characteristics when in coordination with transition metals compared with their well-known five-membered NHC counterparts.44–47 Simultaneously, MIC-metal complexes were found not only to exhibit extraordinary catalytic ability for a variety of chemical transformation reactions,48 but also to have a wide range of applications in photophysics,49 medicinal chemistry, and many other fields.50,51 Although the number of MIC-metal complexes has increased rapidly during the past decade, only a few such complexes featuring mono- or bidentate MIC ligands with attractive optical features have been demonstrated to date.44,52,53 Highly emissive materials based on poly-MIC supramolecular assemblies are relatively underdeveloped, and this is why the luminescence and biological applications of discrete MIC-based supramolecular organometallic assemblies are unknown. Inspired by the attractive AIE performance of the TPE unit and the improved stability features of MIC-metal complexes, we became interested in studying mesoionic carbene-decorated tetraphenylethene (MIC-TPE) derivatives, with the aim of obtaining MIC-based fluorescent organometallic cages that can be used in fluorescence sensing and bioimaging applications. Herein, we designed and synthesized two fluorescent MIC organometallic assemblies [M4( 2)2](OTf)4 (M = Ag and Au) and the MIC-AuI complex Au4( 2)Cl4 by employing a new tetra-dentate MIC-TPE ligand precursor featuring four 1,2,3-triazolium moieties on a TPE core scaffold. These complexes were comprehensively characterized by multinuclear NMR spectroscopy, high-resolution electrospray ionization mass spectrometry (HR-ESI MS), and X-ray diffraction analyses. Compared with the tetradentate 1,2,3-triazolium salt [H4( 2)](OTf)4, the organometallic cages [M4( 2)2](OTf)4 (M = Ag and Au) show strong fluorescence in dilute solutions ascribed to the formation of MI–CMIC bonds, which restricted the rotation of the phenyl rings and pendant triazolium groups, and any twisting of the double bond. In addition, these organometallic cages not only showed tunable fluorescence wavelength and intensity behaviors in mixed solvent systems and at various temperature while in solution, but also exhibited pressure-induced, red-shifted, and decreased emissions in the solid state with gradually increasing external pressure. Furthermore, the MIC-AgI/AuI cages [M4( 2)2](OTf)4 were used in cell imaging, and demonstrated effective suppression of cancer cell growth. To the best of our knowledge, this represents the first example of MIC-AgI/AuI cages showing a fluorescence response to external pressure stimuli and their application in the imaging and treatment of cancer cells. Experimental Methods All the reactions were carried out under nitrogen atmosphere using standard Schlenk techniques. Solvents were freshly distilled by standard procedures prior to use. All chemical reagents were purchased from commercial sources and used without further purification. 1H, 13C{1H}, and two-dimensional (2D) NMR spectra were recorded on AVANCE III 400 or AVANCE III 600 (Bruker, Switzerland) spectrometers. Chemical shifts (δ) are expressed in ppm downfield from tetramethylsilane (TMS) using the residual protonated solvent as an internal standard (CDCl3 = 7.26 ppm, (CD3)2SO = 2.50 ppm, CD3CN = 1.94 ppm, or TMS = 0 ppm). Coupling constants are expressed in Hertz. HR-ESI MS experiments were performed with a Bruker Daltonics micrOTOF-Q II mass spectrometer (Bruker Daltonics Corp., Bremen, Germany) in ESI mode. The UV–vis experiments were conducted on an Agilent Cary-100 spectrophotometer (United States). The fluorescence experiments were performed on a QuantaMaster 8000 spectrometer (slit width: 2 nm; HORIBA Scientific, Canada). Fluorescence decay profiles were recorded on an FLS920 spectrometer (Edinburgh Instruments, Livingston, United Kingdom). The experimental quantum yields were determined by recording the emission signals within an integrating light sphere on a QuantaMaster 8000 photoluminescence spectrometer equipped with an ozone-free Xenon Arc Lamp (75 W) and photomultiplier R928P (Hamatsu Photonics, Japan). Experimental details for all new compounds and organometallic assemblies, including synthesis, characterization, crystal diffraction data (CIF), photophysical properties, high-pressure photoluminescence studies, and cell imaging and anticancer study, are available in the Supporting Information. Results and Discussion Synthesis and molecular structure To synthesize the MIC-TPE ligand precursor [H4( 2)](OTf)4, we employed a stepwise synthesis strategy. First, a copper-catalyzed 1,3-cycloaddition reaction (CuAAC) between 1,1,2,2-tetrakis(4-ethynylphenyl)ethene and 2-azido-1,3,5-trimethylbenzene generated the key compound 1,1,2,2-tetrakis(4-(1-mesityl-1H-1,2,3-triazol-4-yl)phenyl)ethene ( 1) in 81% yield ( Supporting Information Scheme S1 and Figures S1, S2, and S16). Subsequent alkylation to produce the tetra-1,2,3-triazolium salt was accomplished with methyl trifluoromethanesulfonate, which afforded [H4( 2)](OTf)4 as a colorless solid in an excellent yield of 92%. The salt [H4( 2)](OTf)4 was characterized by NMR spectroscopy (1H, 13C{1H}) and HR-ESI MS. The 1,2,3-triazolium C–H resonance appeared at δ = 9.42 ppm in the 1H NMR spectrum ( Supporting Information Figure S3). The 13C NMR spectrum showed the resonance of the C–H carbon of the triazolium rings at δ = 145.1 ppm ( Supporting Information Figure S4). HR-ESI (m/z): [H4( 2)](OTf)4 calcd for {[H4( 2)](OTf)2}2+, 715.2673; found, 715.2581; calcd for {[H4( 2)](OTf)}3+, 427.1940; found, 427.1897. The peaks were isotopically resolved and are in complete agreement with the theoretical distribution ( Supporting Information Figure S17). The reaction of MIC-TPE tetrakis(1,2,3-triazolium) salt [H4( 2)](OTf)4 with an excess of Ag2O in CH3CN at 80 °C for 24 h with exclusion of light resulted in the formation of the prism-like MIC-AgI organometallic cage [Ag4( 2)2](OTf)4 in 67% yield (Scheme 1). The formation of a tetra-nuclear structure was confirmed by NMR spectroscopy (1H, 13C{1H}, and 2D NMR) and HR-ESI MS ( Supporting Information Figures S5–S8 and S18). The 1H NMR spectrum of the complex no longer exhibited the resonance attributed to the C1 proton of the parent triazolium salt ( Supporting Information Figure S5). It is worth noting that the resonances of H10 and H12 on the peripheral mesitylene group of the tetrakis(1,2,3-triazolium) ligand were split into two sets of signals, which indicated different chemical environments for the pairs (H10 and H12) and (H14 and H15) resulting from restricted rotation of the mesitylene groups within the organometallic cage [Ag4( 2)2](OTf)4 (Scheme 1 and Supporting Information Figure S5). HR-ESI (m/z): [Ag4 2)2](OTf)4 calcd for {[Ag4( 2)2](OTf)2}2+, 1494.3640; found, 1494.3020; calcd for {[Ag4( 2)2](OTf)}3+, 946.5924; found, 946.5624 ( Supporting Information Figure S18). All these results confirm a successful preparation of the MIC-AgI cage. Scheme 1 | TPE-bridged 1,2,3-triazolylidene MIC precursor and its organometallic assemblies. Download figure Download PowerPoint The composition and coordination geometry of the molecular [Ag4( 2)2](OTf)4 was further established by X-ray diffraction studies. A colorless crystal was obtained by slow diffusion of diethyl ether into the saturated CH3CN solution of the compound [Ag4( 2)2](OTf)4 in the presence of NH4PF6 at ambient temperature. The structure analysis confirmed the formation of a prism-like tetrasilver octacarbene complex wherein the two TPE-bridged tetra-MIC ligands are flanked by four linear silver(I) ions (Figure 1a). Figure 1 | (a) Molecular structure of the [Ag4(2)2]4+ cation in [Ag4(2)2](PF6)4. (b) Top view of [Ag4(2)2]4+ cation. Color code: C, grey; Ag, pink; N, blue. Hydrogen atoms and counterions omitted for clarity. Download figure Download PowerPoint The metric parameters found in the [Ag4( 2)2]4+ cation [Ag–CMIC, 2.073(5)–2.082(4) Å; CMIC–Ag–CMIC, 179.49(18)–179.66(19)°] are in good agreement with the values previously described for a linear AgI(MIC)2 complex.54 The four silver atoms are oriented in a slightly twisted rectangle featuring two short [Ag1–Ag2*, 8.488 Å; Ag1*–Ag2, 8.488 Å] and two long [Ag1–Ag2, 11.375 Å; Ag1*–Ag2*, 11.375 Å] Ag•••Ag separations in [Ag4( 2)2]4+ (Figure 1b). The phenyl groups of the TPE ligand are rotated out of the ligand-plane to minimize steric repulsion (Figure 1). The neutral 1,2,3-triazolylidene gold(I) chloride complex Au4( 2)Cl4 was synthesized from the MIC-TPE precursor [H4( 2)](OTf)4 and Ag2O in the presence of NMe4Cl in a 1:1 solvent mixture of dichloromethane and CH3CN; subsequent transmetalation with AuCl(tht) (tht = tetrahydrothiophene) in dichloromethane resulted in Au4( 2)Cl4 in 70% yield (Scheme 1). The absence of the signal at δ = 9.42 ppm attributed to the triazolium C–H resonance of [H4( 2)](OTf)4 in the 1H NMR spectrum and the appearance of a new signal at δ = 160.2 ppm in the 13C{1H} NMR spectrum indicated complete degradation of 1,2,3-triazolium and the formation of CMIC–AuI bonds ( Supporting Information Figures S9–S11). The HR-ESI (m/z): Au4( 2)Cl4 calcd for [Au4( 2)Cl3]+, 2023.3701; found, 2023.3407; calcd for [Au4( 2)Cl2]2+, 993.2016; found, 993.1626, was consistent with the theoretical isotopic distribution ( Supporting Information Figure S19). The MIC-AuI cage [Au4( 2)2](OTf)4 was prepared by transmetalation of the supramolecular assembly derived from the corresponding triazolylidene MIC-AgI cage with AuCl(tht) in 67% yield (Scheme 1). The [Au4( 2)2](OTf)4 complex was characterized by NMR spectroscopy and HR-ESI MS ( Supporting Information Figures S12–S15 and S20). The 13C NMR data showed a downfield Au-CMIC signal at δ = 173.0 ppm ( Supporting Information Figure S13), in agreement with data previously reported for a [AuI(MIC)2] complex.54 Alternatively, [Au4( 2)2](OTf)4 could also be obtained from MIC-AuI complex Au4( 2)Cl4 in the presence of AgOTf in dichloromethane solution (Scheme 1). Photophysical properties Steady-state absorption and fluorescence emission spectra for the tetrakis(1,2,3-triazolium) salt [H4( 2)](OTf)4 and its metal complexes in CH3CN are shown in Figure 2. In the UV–vis absorption spectra, the MIC-metal complexes of [M4( 2)2](OTf)4 (M = Ag and Au) and Au4( 2)Cl4 showed very similar absorption behavior with vibronic-structured absorption bands in the range of 250–360 nm. Compared with the tetrakis(1,2,3-triazolium) salt [H4( 2)](OTf)4, a slight red-shift was observed upon the formation of metal carbene complexes of Au4( 2)Cl4 and [M4( 2)2](OTf)4 (M = Ag and Au). In addition, the organometallic cages of [M4( 2)2](OTf)4 (M = Ag and Au) display obviously enhanced vibrational structure in the UV–vis absorption spectra, which is indicative of increased rigidity occurring upon triazolylidene coordination (Figure 2a). Figure 2 | (a) UV–vis absorption spectra, (b) fluorescence spectra (c = 10.0 μM, λex = 334 nm), and (c) fluorescence images (upon excitation at 365 nm using a UV lamp, c = 10.0 μM) of [H4(2)](OTf)4, [M4(2)2](OTf)4 (M = Ag and Au), and Au4(2)Cl4 in CH3CN at 298 K. (d) Fluorescence quantum yields of [H4(2)](OTf)4, [M4(2)2](OTf)4 (M = Ag and Au), and Au4(2)Cl4 in CH3CN. Download figure Download PowerPoint In the fluorescence emission spectra, tetrakis(1,2,3-triazolium) salt [H4( 2)](OTf)4 is weakly emissive (ФF < 1%) (Figures 2band 2d) in CH3CN because of the prevalence of nonradiative decay resulting from intramolecular free-rotation of the phenyl rings and pendant triazolium groups and twisting of the double bonds. Upon formation of complex Au4( 2)Cl4, the fluorescence spectrum showed a moderate emission band centered at ∼490 nm. In sharp contrast, [M4( 2)2](OTf)4 (M = Ag and Au) are strongly emissive at ∼514 nm and a bright green fluorescence (ФF up to 32%) was observed in CH3CN. This is attributed to the locking of nonemissive tetrakistriazolylidene ligands within the rigid discrete organometallic cages (Figures 2a and 2c and Supporting Information Figures S21 and S22 and Table S2). Moreover, the fluorescence intensity of [Au4( 2)2](OTf)4 was observed to be slightly weaker compared with [Ag4( 2)2](OTf)4 due to the heavy atom effect.15–17,55,56 The fluorescence lifetimes of tetrakis(1,2,3-triazolium) salt and its metal complexes are on the scale of nanoseconds both in CH3CN solution and in the solid state, which suggests a typical fluorescent emission behavior ( Supporting Information Table S2 and Figures S37–S42).28 Compared with TPE-based metal–organic frameworks (MOFs), which are generally insoluble,57–60 the enhanced solubility of these TPE-based supramolecular metallacages is advantageous for the investigation of their fluorescent properties in various solutions.61–64 The organometallic cage [Au4( 2)2](OTf)4 exhibited good photophysical properties in various solvent systems by UV–vis and fluorescence experiments (Figure 3 and Supporting Information Figure S23 and Table S1). The UV–vis spectra of [Au4( 2)2](OTf)4 in both polar [e.g., dimethyl sulfoxide (DMSO), dimethylformamide (DMF), MeOH, CH3CN, and ethyl acetate (EA)] and nonpolar solvents [e.g., CHCl3, CH2Cl2 and tetrahydrofuran (THF)] showed similar absorption bands approximately in the range of 250–360 nm ( Supporting Information Figure S23 and Table S1). The solution of [Au4( 2)2](OTf)4 also showed a relatively stable intensity and fluorescence quantum yield (ФF = 22–32%) with a bright green maximum emission centered from 510 to 520 nm in different solvent systems (Figures 3a–3c and Supporting Information Table S1). Figure 3 | (a) Fluorescence spectra (λex = 334 nm, c = 10.0 μM) of [Au4(2)2](OTf)4 in various solvents and (b) fluorescence quantum yields of [Au4(2)2](OTf)4 in different solvents. (c) Fluorescence images of [Au4(2)2](OTf)4 upon excitation with a UV lamp (λex = 365 nm). Download figure Download PowerPoint Solvatochromic behaviors and thermal response properties MIC-AuI complex Au4( 2)Cl4 and organometallic cages [M4( 2)2](OTf)4 (M = Ag and Au) are generally stable for over two weeks in both solution phase and in the solid state, which allows exploration for further applications. To probe the AIE properties of these MIC-metal complexes, the fluorescence emission of Au4( 2)Cl4 in dichloromethane/hexane solutions with varying hexane content was recorded ( Supporting Information Figures S24 and S25). Au4( 2)Cl4 displayed relatively weak fluorescence and low emission intensity in mixed solutions where the hexane content was <50% ( Supporting Information Figure S25). However, when the hexane content was increased to 60%, the emission maximum of Au4( 2)Cl4 underwent a sudden and large blue shift from 521 to 491 nm. The largest blue shift (∼52 nm) was observed when the hexane content was increased to 70%. However, further increase of the hexane content from 80 to 90%, abruptly amplified the fluorescence intensity ( Supporting Information Figure S25). The exhibited AIE behavior with the increase of hexane content is attributed to the limitation of intramolecular rotation due to the formation of aggregates. At the same time, the increase in the hexane content creates a less polar environment which can attenuate the charge-transfer (CT) process. The CT process can red shift the emission color and decrease the emission efficiency of AIE chromophores in a polar solution. In addition, metal-to-ligand CT (MLCT) processes with the assemblies may also be suppressed by a less-polar environment. Therefore, it is reasonable that the emission peak of the complex Au4( 2)Cl4 blue-shifted with the increase of hexane content.24,25 The fluorescence response performance of organometallic cage [Ag4( 2)2](OTf)4 in CH3CN/H2O mixtures with different H2O contents was also monitored ( Supporting Information Figure S26). When the water content was <50%, the emission spectra were almost unchanged except for a slight increase in the fluorescence intensity. When the water content was increased gradually from 60% to 90%, the emission maximum underwent a continuous blue shift (∼25 nm) and the emission color changed from green to cyan ( Supporting Information Figures S26 and S27). The above results confirm that the fluorescence emission peaks and intensities of the MIC-metal complexes can be tuned. Furthermore, the fluorescence emission profiles of [M4( 2)2](OTf)4 (M = Ag and Au) with respect to stimulation by temperature were also investigated. Upon heating from −20 to 80 °C, the emission intensities of [M4( 2)2](OTf)4 (M = Ag and Au) exhibited an approximately linear and gradual weakening, but no obvious temperature effect on the emission wavelength was observed ( Supporting Information Figures S28 and S29). Moreover, emission intensities recovered when the complexes were re-cooled, indicating the reversibility of any fluorescence behaviors due to temperature effects. Mechanochromic properties To explore the effect of external pressure on the fluorescence emission in the solid state, fluorescence spectra and images were collected at high pressure using the diamond anvil cell (DAC) technique. MIC-AuI complex Au4( 2)Cl4 and organometallic cage [Au4( 2)2](OTf)4 showed a bright-green emission with maximum emission at approximately 492 and 508 nm, respectively, under ambient conditions (Figures 4a and 4b). During the compression process, the maximum fluorescence emission of Au4( 2)Cl4 and [Au4( 2)2](OTf)4 showed a large red shift to 590 nm at 10.59 GPa and to 589 nm at 12.20 GPa, respectively. Meanwhile, the fluorescence intensity of Au4( 2)Cl4 gradually decreased until effective quenching as the pressure increased to 9.65 GPa (Figure 4a). The phenomenon that the complexes show red shift in emission wavelength and decrease in emission intensity after applying external pressure is probably caused by the planarization of benzene rings in TPE that promotes tighter intermolecular packing under external mechanical pressure.7,33 However, the fluorescence intensity of [Au4( 2)2](OTf)4 decreased more slowly when compared with Au4( 2)Cl4 upon gradually increasing applied pressure. Fluorescence was not quenched completely even at 12.20 GPa, which is evident in the fluorescent images depicted in Figures 4c and 4d. Such subtle differences in fluorescence response under applied pressure are attributed to the formation of CMIC–Au–CMIC bonds and the presence of noncoordinating anions, which would suppress any additional intra- and intermolecular packing between the two MIC ligands arising from the same or different organometallic cages. When the external pressure was gradually withdrawn, the fluorescence intensity of both Au4( 2)Cl4 and [Au4( 2)2](OTf)4 recovered to their initial states, and the corresponding emission wavelength blue-shifted to the original emission wavelength ( Supporting Information Figures S31 and S32). These results further support the fact that the conformational planarization of benzene rings in TPE and intermolecular tighter packing are the main reasons for the red shift in emission wavelength and decrease in emission intensity.7,33 Similarly, the emission spectra of [Ag4( 2)2](OTf)4 at increasing applied pressures exhibited a red shift in emission wavelength and continuously decreased in intensity ( Supporting Information Figure S30). Notably, similar mechanochromic responses have also been observed in TPE-based MOFs and SCCs systems.7,33,65 Figure 4 | Emission spectra of (a) Au4(2)Cl4 and (b) [Au4(2)2](OTf)4 under applied external pressures. Corresponding fluorescent images of (c) Au4(2)Cl4 and (d) [Au4(2)2](OTf)4. Download figure Download PowerPoint Cell imaging and anticancer study Although the cell imaging and anticancer abilities of TPE-based SCCs have been intensively studied,6,66,67 the application of TPE-based organometallic cages in this field has not been reported to date. We were inspired by previous examples of NHC-AuI complexes used in cell imaging and anticancer applications.68–73 Considering that the fluorescence behavior and stability of MIC-AuI complex and organometallic cages [M4( 2)2](OTf)4 (M = Ag and Au) are significantly improved, we report the first confocal laser scanning microscopy (CLSM) studies of organometallic cages [M4( 2)2](OTf)4 (M = Ag and Au) clearly demonstrating cell imaging ability. These studies used three different cancer cell lines representing different types of cancer: A549 (nonsmall cell lung cancer), HeLa (cervical cancer), and SMMC-7721 (hepatocellular cancer). The human normal hepatocyte cells (L-02) were used as control. Briefly, cultures of each cell line were incubated with both organometallic complexes and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil) for 4 h before fluorescent imaging. As illustrated in Figure 5, the bright blue fluorescent images derived from [Au4( 2)2](OTf)4 were observed to overlap with the cytoplasm imaged by Dil (red fluorescence), which suggests that fluorescent organometallic cages can be used as contrast agents for cell imaging. Figure 5 | CLSM images of A549, HeLa, SMMC-7721, and L-02 cells incubated with cell membrane dye, Dil (red), and organometallic cage [Au4(2)2](OTf)4 (2 μM) (blue) for 4 h. Scale bars represent 20 μm. Download figure Download PowerPoint In addition, the cell imaging results of Au4( 2)Cl4 and [Ag4( 2)2](OTf)4 show that they both have potential as bioimaging agents, but the fluorescence intensity and cell uptake efficiency are lower than [Au4( 2)2](OTf)4 ( Supporting Information Figures S33 and S34). The same cell imaging experiments were also performed in human normal hepatocyte (L-02) cells, and the results show that only weak blue fluorescence and a negligible colocalization ratio (<10%) were observed in L-02 cells, indicating that they are highly specific for cancer cell imaging (Figure 5 and Supporting Information Figures S33–S35). Finally, the antiproliferative activities of MIC-AuI complex Au4( 2)Cl4 and organometallic cages [M4( 2)2](OTf)4 (M = Ag and Au) against three human cancer (A549, HeLa, and SMMC-7721) and human normal hepatocyte (L-02) cell lines were evaluated via the standard 3-(4′,5′-dimethylthiazol-2′-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Table 1 and Supporting Information Figure S36). As shown in Table 1, the organometallic cages [Ag4( 2)2](OTf)4 and [Au4( 2)2](OTf)4 show considerable anticancer activity with the half maximal inhibitory concentration (IC50) values ranging from 2.