Red-Emissive Organic Room-Temperature Phosphorescence Material for Time-Resolved Luminescence Bioimaging

Open AccessCCS ChemistryCOMMUNICATION5 Aug 2022Red-Emissive Organic Room-Temperature Phosphorescence Material for Time-Resolved Luminescence Bioimaging Wenbo Dai†, Yahui Zhang†, Xinghui Wu, Shuai Guo, Jieran Ma, Jianbing Shi, Bin Tong, Zhengxu Cai, Haiyan Xie and Yuping Dong Wenbo Dai† Beijing Key Laboratory of Construction Tailorable Advanced Functional Materials and Green Applications, School of Materials Science and Engineering, Beijing Institute of Technology, Beijing 100081 †W. Dai and Y. Zhang contributed equally to this work.Google Scholar More articles by this author , Yahui Zhang† School of Life Science, Advanced Research Institute of Multidisciplinary Science, Beijing Institute of Technology, Beijing 100081 †W. Dai and Y. Zhang contributed equally to this work.Google Scholar More articles by this author , Xinghui Wu Beijing Key Laboratory of Construction Tailorable Advanced Functional Materials and Green Applications, School of Materials Science and Engineering, Beijing Institute of Technology, Beijing 100081 Google Scholar More articles by this author , Shuai Guo Beijing Key Laboratory of Construction Tailorable Advanced Functional Materials and Green Applications, School of Materials Science and Engineering, Beijing Institute of Technology, Beijing 100081 Google Scholar More articles by this author , Jieran Ma Beijing Key Laboratory of Construction Tailorable Advanced Functional Materials and Green Applications, School of Materials Science and Engineering, Beijing Institute of Technology, Beijing 100081 Google Scholar More articles by this author , Jianbing Shi Beijing Key Laboratory of Construction Tailorable Advanced Functional Materials and Green Applications, School of Materials Science and Engineering, Beijing Institute of Technology, Beijing 100081 Google Scholar More articles by this author , Bin Tong Beijing Key Laboratory of Construction Tailorable Advanced Functional Materials and Green Applications, School of Materials Science and Engineering, Beijing Institute of Technology, Beijing 100081 Google Scholar More articles by this author , Zhengxu Cai *Corresponding author: E-mail Address: email protected Beijing Key Laboratory of Construction Tailorable Advanced Functional Materials and Green Applications, School of Materials Science and Engineering, Beijing Institute of Technology, Beijing 100081 Google Scholar More articles by this author , Haiyan Xie School of Life Science, Advanced Research Institute of Multidisciplinary Science, Beijing Institute of Technology, Beijing 100081 Google Scholar More articles by this author and Yuping Dong Beijing Key Laboratory of Construction Tailorable Advanced Functional Materials and Green Applications, School of Materials Science and Engineering, Beijing Institute of Technology, Beijing 100081 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.021.202101120 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Organic room-temperature phosphorescence (RTP) materials have been used in high-resolution imaging. However, the development of long-wavelength-emission RTP materials in aqueous solution remains a challenge. Here, we report red-emissive RTP materials via integration of the ring-fusing effect and host–guest interaction. The fused-ring guest molecule showed a low triplet state for long-wavelength emission, and the host molecule can bridge the huge energy gap between the triplet and singlet state of guest molecules for highly efficient intersystem crossing processes. The fused-ring host–guest material showed red emission with a long lifetime up to 274 ms because of enhanced conjugation and restriction of intramolecular motion. The well-dispersed phosphorescent nanoparticles (NPs) with a uniform size of approximately 200 nm show good biocompatibility and low cytotoxicity. The NPs are applied to time-resolved luminescence imaging to eliminate background fluorescence interference in vitro and in vivo. Download figure Download PowerPoint Introduction The development of time-resolved luminescence imaging (TRLI) has allowed researchers to evolve pure organic luminophores that feature long-lived room-temperature phosphorescence (RTP)1–9 or afterglow luminescence10–12 as probe signal primitives. By taking advantage of the considerably longer relaxation time in phosphorescence compared to fluorescence,2,13 TRLI avoids influencing biological autofluorescence and background interference in detection and imaging processes.14,15 Furthermore, long-wavelength phosphorescent emission reduces the self-absorption of biological tissues and scattering of photons,3,16–18 thereby increasing penetration depth, signal-to-noise ratio, and imaging resolution for in vivo imaging.3,17,19,20 Moreover, organic phosphor without toxic heavy metals possesses excellent biocompatibility.21–23 Therefore, the development of organic RTP materials with long-wavelength emission and long lifetime is of great significance to the construction of phosphorescent probes and realization of biological TRLI. Despite great success in molecular design engineering of pure organic phosphorescent materials with colorful phosphorescent emission,19,24–28 it remains a formidable challenge to obtain highly efficient red phosphorescence under ambient conditions due to intense nonradiative transitions of triplet excitons, such as self-quenching by π–π stacking, molecular rotations and variations, and inefficient intersystem crossing (ISC).29–33 This limits potential applications of pure organic phosphorescence in biological fields. Impressively, Wu et al.16 developed self-assembled organic nanoparticles (NPs) from difluoroboron β-diketonate compounds in aqueous solution. The NPs showed bright RTP, effective uptake, and bright imaging of HeLa cells under both visible- and near infrared-light excitation. Huang et al.34 proposed a versatile approach based on hollow mesoporous silica NPs for the realization of hydrophilic dispersion of hydrophobic organic phosphors at nanoscale and demonstrated their potential for in vivo phosphorescence imaging. However, these materials rely on a donor–acceptor (D–A) structure that facilitates strong intramolecular charge transfer (ICT) promoting long-emission wavelength but suffers from low efficiency and short lifetime.16,35 Therefore, it is imperative to develop a new molecular design strategy to obtain long-lifetime and high-efficiency red-emissive phosphorescent materials. Fused-ring molecules can be potential phosphorescent molecules with long-emission wavelength. The triplet-state energy level of fused-ring molecules can be half of their singlet-state, resulting in a narrow triplet bandgap and long-wavelength triplet emission.36–40 However, the large energy gap between the lowest singlet and triplet state (ΔEST) prevents highly efficient ISC to realize phosphorescence at room temperature.13,28,41–43 Recently, our group24 developed a novel method to generate RTP by embedding guest materials into a rigid or crystalline small molecular host matrix. The host matrix not only provides a rigid environment to restrict movement, but also serves as a bridge to facilitate ISC between singlet and triplet states of the guest.24,44–49 Herein, we propose a strategy to achieve highly efficient red-phosphorescence emission via the combination of molecular engineering and a host–guest system (Scheme 1). Two methylene groups were introduced into 1,2,5-triphenyl-1H-pyrrole ( TPPy) to lock its molecular conformation and extend conjugation (Scheme 1a). Single crystal analysis demonstrated 5-phenyl-10,11-dihydro-5H-diindeno1,2-b:2′,1′-dpyrrole ( PDPy) has high molecular planarity, and theoretical calculations indicated that this planer structure could effectively decrease the triplet-state energy level. Triphenylamine ( TPA) was chosen as the host due to its characteristic low melting point and suitable triplet-state energy level. The low melting point facilitated the preparation of large quantities of doped materials by the melt-casting method, while the suitable triplet energy level was used to bridge the huge gap between the triplet- and singlet-states of guest molecules for a highly efficient ISC process. Therefore, phosphorescence emission was achieved by doping TPPy and PDPy as guests into TPA (Scheme 1b). Intriguingly, PDPy/TPA doped materials exhibited a long phosphorescence wavelength (λP) of 606 nm, as well as a long lifetime of 274 ms. Such a simple guest molecule achieved red-phosphorescent emission, which has been rarely reported. Moreover, well-dispersed organic phosphorescence NPs were prepared by a top-down approach with particle size of approximately 230 nm and long emission lifetime of 139 ms in aqueous media. Given the good biocompatibility and long-lived emission of the phosphorescent NPs, dual-channel and millisecond-level TRLI in vitro and in vivo were conducted. Scheme 1 | Design concept for highly efficient red-phosphorescence material through (a) molecular engineering and (b) host–guest system. Download figure Download PowerPoint Experimental Methods Materials and characterization The host and guest molecules were purified by column chromatography twice, followed by recrystallization. 1H and 13C NMR spectroscopy was carried out on a Bruker ARX400 spectrometer (Bruker, Germany) with CDCl3 as the solvent. Mass spectra were obtained with a Bruker BIFLEX III mass spectrometer (Bruker, Germany). UV–vis absorption spectra were measured by Persee TU-1901 spectrophotometer (PERSEE, China). Fluorescence and phosphorescence spectra were measured by using a Nanolog FL3-2iHR (Horiba Jobin Yvon, France) spectrophotometer. X-ray crystal structure analysis was performed on a Bruker-AXS SMART APEX2 CCD diffractometer (Bruker AXS, Germany). Powder X-ray diffraction (PXRD) data were measured by Smartlab SE (Rigaku Corporation, Japan). Solid-state emission quantum yields (Φ) were collected by using a FluoroMax-4 (Horiba Jobin Yvon) spectrofluorometer equipped with an integrated sphere. Photoluminescence (PL) time-resolved decays were measured with a DeltaFlex ultrafast lifetime spectrofluorometer (HORIBA Scientific, Japan). Animals studies Six-week-old BALB/c nude mice were purchased from the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animal studies were performed in accordance with the Regulations for Care and Use of Laboratory Animals and Guideline for Ethical Review of Animals (China, GB/T 35892-2018) and the overall project protocols were approved by the Animal Ethics Committee of Beijing Institute of Technology. The accreditation number is BIT-EC-SCXK (Jing) 2019-0010-M-2020019 promulgated by the Animal Ethics Committee of Beijing Institute of Technology. All animals were submitted to controlled temperature conditions (22–26 °C), humidity (50–60%), and light (12 h light/12 h dark, 15–20 lx). They had access to water and food ad libitum in a barrier system from the Beijing Institute of Technology (SYXK (Jing) 20170013). Results and Discussion Compounds TPPy and PDPy were synthesized, and the molecular structures were confirmed by mass spectroscopy (MS) and NMR spectroscopy ( Supporting Information Figures S17–S22 see Supporting Information for details). Both compounds showed good solubility in common organic solvents. Their purities were determined by elemental analysis. Single crystals of the compounds were analyzed by single-crystal X-ray diffraction. UV–vis absorption and PL spectra of TPPy and PDPy were recorded in tetrahydrofuran (THF) solution and the solid state. Compared with TPPy, the intramolecular conjugation of PDPy was effectively extended by the phenyl fused with the pyrrole ring at the 2,5-position, thus red-shifting absorption and fluorescence wavelengths to the long-wavelength region in both solution and solid state (Figures 1a and 1b). The molar extinction coefficient (ε) of ring-fused molecule PDPy was double that of TPPy due to good planarity of the pyrrole backbone (Figure 1a). A high ε favors probe materials for bioimaging.50 Unlike TPPy with the aggregation-induced quenching character,51,52 PDPy showed high luminous efficiencies in both solution and solid state, revealing a dual-phase emission characteristic ( Supporting Information Table S1). Additionally, the emission wavelengths of both compounds showed only a red shift of ca. 10 nm from non-polar hexane to highly polar acetonitrile (MeCN), indicating the absence of solvatochromic properties due to lack of intramolecular D–A interaction ( Supporting Information Figure S1),53 which ensures high luminescence efficiency. Figure 1 | (a) UV–vis absorption and normalized fluorescence spectra of TPPy and PDPy in THF (concentration: 1 × 10−5 mol/L). (b) Normalized absorption and fluorescence spectra of TPPy and PDPy in the solid state. (c) The calculated HOMO and LUMO energy levels of TPPy and PDPy. (d) Diagrams of the TD-DFT calculated energy levels and SOC constants of TPA, TPPy, and PDPy. ε, molar extinction coefficient. Download figure Download PowerPoint TPPy and PDPy were crystallized via slow evaporation under ambient conditions in CH2Cl2/CH3OH (1∶1) solvent. X-ray crystallographic analysis showed that both compounds possess the monoclinic system and are stacked in parallel in space ( Supporting Information Tables S2 and S3). TPPy adopted a highly twisted conformation because observed torsion angles between the pyrrole group and adjacent phenyl rings are 39.04°, 69.14°, and 39.04°, respectively ( Supporting Information Figure S2). In contrast, the conjugated backbone of PDPy was fully coplanar. The fused core with high planarity effectively extended the conjugation and enhanced the emissive intensity of the crystalline state ( Supporting Information Figure S3). Moreover, the phenyl ring at the 1-position with dihedral angles larger than 50° avoids the detrimental close packing and enhances the quantum yields in the solid state.53 Density functional theory (DFT) calculations were carried out on the basis set of B3LYP/6-311G (d) via Gaussian 09 (see details in Supporting Information).54 The frontier molecular orbitals and the energy gaps (Eg) of TPPy and PDPy are shown in Figure 1c. The electron clouds of the highest occupied molecular orbital (HOMO) were predominantly localized on the pyrrole and fused phenyl ring on the 2,5-position of the pyrrole, and the lowest unoccupied molecular orbital (LUMO) was distributed throughout the whole molecule. Hence, no obvious ICT was observed upon excitation, which was responsible for no solvent effects. The electronic clouds of HOMO and LUMO had no obvious separation, resulting in a large ΔEST. Time-dependent DFT (TD-DFT) calculations were performed to calculate the singlet and triplet energy levels of both compounds.55,56 As shown in Figure 1d, the triplet state (T1) of PDPy was obviously lower than that of TPPy, suggesting that the fused ring could effectively lower the triplet energy level, leading to long wavelength phosphorescence emission. However, the large ΔEST and small spin-orbit coupling (SOC) constant did not facilitate highly efficient ISC for phosphorescence at room temperature ( Supporting Information Tables S4 and S5).13 Therefore, RTP could be only achieved via a host–guest doping system. TPPy and PDPy as guests exhibited afterglow in low-temperature solutions (Figures 2a and 2b), but a particularly weak phosphorescent emission in the crystalline state at 77 K ( Supporting Information Figure S4). The low-temperature phosphorescence wavelength of PDPy was longer than that of TPPy, which was consistent with theoretical calculations ( Supporting Information Table S6). The prompt and delayed spectra of TPA was also measured in 2-MeTHF solution at 77 K to calculate the energy gap between S1 and T1 ( Supporting Information Figure S5). The T1 energy level of the host lies between the T1 and S1 energy levels of the guests, thereby bridging the huge gap between the triplet and singlet states of guest molecules for a highly efficient ISC process. Figure 2 | (a) Prompt (blue line) and delayed emission (yellow line) spectra of TPPy in 2-MeTHF at 77 K (top, concentration: 1 × 10−5 mol/L) and TPPy/TPA crystalline materials (bottom) (λex.Prompt: 320 nm; λex.Delayed: 360 nm; delayed time: 50 μs). (b) Prompt (blue line) and delayed emission (red line) spectra of PDPy in 2-MeTHF at 77 K (top, concentration: 1 × 10−5 mol/L) and PDPy/TPA crystalline materials (bottom) (λex.Prompt: 320 nm; λex.Delayed: 360 nm; delayed time: 50 μs). (c) UV–vis absorption spectra of the doped materials and individual guest and host in the solid state. (d) Phosphorescence decay curves of TPPy/TPA and PDPy/TPA crystalline materials. (e) The photographs of TPPy/TPA (left) and PDPy/TPA (right) crystals at different time intervals under 365 nm UV light. Download figure Download PowerPoint Encapsulation of the luminophore in the matrix avoided phosphorescence quenching caused by the presence of water and oxygen, while increasing intermolecular interaction to suppress nonradiative decay.24,57–61 The two host–guest doping materials in a molar ratio of 1∶1000 were prepared by the melt-casting method. Under UV irradiation, TPPy/TPA crystals exhibited blue emission with PL peak at 384 nm (Figure 2a). After removal of irradiation, a yellow afterglow lasted for several seconds (Figure 2e); its maximum at 567 nm (Figure 2a) and lifetime of 122 ms (Figure 2d) verified RTP. Notably, the introduction of two methylene groups showed orange-red phosphorescence of PDPy/ TPA (Figure 2e). Delayed spectra analysis of PDPy/ TPA crystals displayed a major peak at 606 nm and shoulder at ca. 670 nm (Figure 2b), along with a long-lived lifetime of 274 ms (Figure 2d). The high energy bands (410 nm) can be attributed to delayed fluorescence, because the delayed fluorescence curves match the fluorescence curves of the doped materials (Figure 2b). Moreover, the lifetime of delayed fluorescence can reach 2.18 ms ( Supporting Information Figure S6). The red-shifted RTP of PDPy/TPA crystals was ascribed to a reduced triplet-state energy caused by the fused-ring. Moreover, the fine phosphorescence peak of PDPy/TPA was similar to that of PDPy at 77 K, whereas the RTP spectra of TPPy/TPA showed a broad peak without fine structure, indicating that PDPy with the fused-ring structure could effectively restrict the intramolecular motion (Figures 2a and 2b).62 Therefore, PDPy/TPA showed higher phosphorescence quantum yield and longer emission wavelength than those of TPPy/TPA (Table 1). Table 1 | Photophysical Properties of Host–Guest Molecules, and Their Doping Materials Sample Fluorescence Phosphorescence λF (nm) ΦF (%) τF (ns) λP (nm) ΦP (%) τP (ms) TPA 382 9.7 2.10 521b — — TPPy 387 1.9 1.06 552b — — PDPy 432 73.7 2.04 595b — — TPPy/TPAa 384 20.1 2.12 567 3.4 122 PDPy/TPAa 407 11.5 1.39 606 4.3 274 aNote: Guest:host = 1:1000 (molar ratio) (λex.Prompt: 320 nm; λex.Delayed: 360 nm; delayed time: 50 μs). bMeasured in 2-MeTHF at 77 K (concentration: 1 × 10−5 mol/L). A series of host–guest materials with doping ratios ranging from 1∶10 to 1∶10,000 were also prepared to further investigate the photophysical properties of doped materials. Notably, even when the concentration of guest was as low as 0.01 mol % (1∶10,000), the host–guest materials still produced obvious visible afterglow ( Supporting Information Figure S7). As the ratio of PDPy increased, the fluorescence emission wavelength of the host–guest materials slightly red shifted ( Supporting Information Figure S7b), likely due to the molecular aggregation of the guest molecules at high concentration. As depicted in Supporting Information Figure S8, with increasing doping ratio, no new absorption peak appears in the long-wavelength region, indicating the absence of intermolecular charge transfer (Figure 2c).63,64 Moreover, the phosphorescence lifetimes of the doped materials were basically unchanged ( Supporting Information Figure S9). Therefore, high concentration of host–guest NPs could be fabricated for in vitro and in vivo time-resolved imaging due to the high light absorption efficiency. For biological imaging, PDPy/TPA was encapsulated by the amphiphilic polymer F127 to obtain good water dispersity and stable NPs via the top-down method ( Supporting Information Figure S10a, see Supporting Information for the fabrication of PDPy/ TPA).21 As shown in Figure 3a, dynamic light scattering (DLS) and transmission electron microscopy (TEM) images revealed that PDPy/TPA NPs were spherical with an average diameter of 232 nm and well-dispersed in aqueous solution. No precipitation of NPs was observed after storage at 4 °C, and only a small change in size occurred after incubation in phosphate-buffered saline (PBS) solution for one week ( Supporting Information Figure S10b). As expected, the fluorescence and phosphorescence spectra of NPs were very similar to those of crystals under ambient condition, with the major emission peaks at ca. 400 and 610 nm, respectively (Figure 3b). Furthermore, the phosphorescence lifetime of NPs reached 139 ms (Figure 3c), demonstrating the long-lived phosphorescence feature. The NPs exhibited a triplet-state-related emission behavior that is sensitive to air at room temperature ( Supporting Information Figure S11). Therefore, the presence of oxygen in the water may decrease the lifetime of the NPs compared to crystalline materials. Moreover, the introduction of the F127 also decreased the crystallinity of the NPs ( Supporting Information Figure S12). Therefore, the non-radiative transition in NPs was not as well suppressed as that in crystalline powder, which was also responsible for the decrease of the emission lifetime and intensity. However, the NPs still have some crystallinity which avoids the complete quenching of the triplet excitons by interactions with water and oxygen. Figure 3 | (a) The particle size distribution for PDPy/TPA NPs (Inset: Representative TEM image of the NPs, scale bar: 500 nm). (b) Normalized absorption, fluorescence, and phosphorescence spectra of NPs in aqueous solution. (c) Phosphorescence decay curves of NPs in aqueous solution. (d) Images of bright-field, blue channel (420–500 nm), red channel (600–680 nm), and merge. (e) Left: Intensity of lifetime decay profile of emission band. The rest: Images of HeLa cells at different delay time. Download figure Download PowerPoint Based on structural design, energy level modulation, and host–guest interaction, the short-wavelength fluorescence emission and long-wavelength phosphorescence emission of PDPy/TPA NPs did not overlap. Therefore, PDPy/TPA NPs possessed excellent dual-channel imaging capabilities.65 HeLa cells are widely used in tumor research and evaluation of biological properties of biomaterials. Therefore, HeLa cells were used for the evaluation of the biological performance of PDPy/TPA NPs. The apoptosis assay experiment with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) is shown in Supporting Information Figure S13. Almost 85% of the HeLa cells survived after 24 h of co-staining at the concentration of 35 μg mL−1. After incubating with NPs (35 μg for 10 dual-channel by microscopy of PDPy/TPA NPs with HeLa cells performed by nm blue and red luminescence was observed in the of the which indicated that NPs could the (Figure The was good for the dual-channel imaging. The blue (420–500 nm) and red (600–680 nm) emission were to the fluorescence and phosphorescence of the NPs, emission from a host–guest system has and than The dual-channel imaging with the design of host–guest NPs provides a new strategy of biological imaging. Furthermore, taking advantage of the long emission lifetime of PDPy/TPA NPs to eliminate the biological background fluorescence, TRLI in cells was carried As shown in Figure and Supporting Information Figure long-lived signal was obtained and from background The image of Figure was the intensity of the lifetime decay profile of emission in HeLa indicating the lifetime intensity distribution of the phosphorescence signal in the this a series of images with different decay were the image with no time from the whole are After a time intense from after the phosphorescence were still PDPy/TPA NPs stable signal that may eliminate interference from The time-resolved was to eliminate the fluorescence interference effectively by the delay time. Therefore, this phosphorescence is for imaging of biological TRLI in HeLa cells of PDPy/TPA NPs the for phosphorescence time-resolved imaging in vivo. The = in vivo imaging imaging system was used to the and the images under In Supporting Information Figure was the UV time for PDPy/TPA NPs. After with a 365 nm UV for the images were time: The shown in Figures and verified that phosphorescent signal from PDPy/TPA NPs could still be even after the removal of light excitation. PDPy/TPA NPs μg were to nude of afterglow intensity of PDPy/TPA NPs in vivo was up to × × (Figures and The ratios reached the advantage of TRLI with background Figure 4 | (a) images after UV of PDPy/TPA NPs μg for (b) of afterglow of in (c) In vivo afterglow imaging of mice after of PDPy/TPA NPs μg images were after UV of mice for (d) of afterglow of in Download figure Download PowerPoint have developed long-wavelength-emission organic NPs based on a fused-ring host–guest system in aqueous solution. The NPs showed RTP, effective uptake, and time-resolved bright imaging in vitro and in vivo. DFT and single-crystal structure analysis the fused-ring effect reduced the triplet state energy level and the host–guest interaction the ISC from the singlet to both resulting in long-lived red-emissive RTP. The simple ring-fusing strategy demonstrated not only a for the triplet state, but also for of the of RTP in host–guest Supporting Information Supporting Information is and and preparation single-crystal and in vivo afterglow imaging, NMR and mass of is no of to Information This was by the of and This was also by Beijing Laboratory for Science The for with photophysical and in vitro imaging. of Organic Phosphorescence