Single-Molecule Phosphorescence Resonance Energy Transfer for NIR Targeted Cell Imaging

Single-Molecule Phosphorescence Resonance Energy Transfer for NIR Targeted Cell Imaging | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Single-Molecule Phosphorescence Resonance Energy Transfer for NIR Targeted Cell Imaging Yu Liu, Xiaolu Zhou, Xue Bai, Heng-Yi Zhang, Li-Hua Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3784778/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Jun, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract A single-molecule phosphorescence resonance energy transfer (PRET) system with a large Stokes shift of 367 nm and near-infrared (NIR) emission is constructed by alkyl-bridged methoxy-tetraphenylethylene-phenylpyridines derivative (TPE-DPY), cucurbit[n]uril (CB[n], n = 7,8), and β-cyclodextrin modified hyaluronic acid (HACD). The experiment results demonstrate that the high binding affinity and various stoichiometric ratios of CB[n] (n = 7, 8) to TPE-DPY not only regulate the topological morphology of supramolecular assembly but also induce different phosphorescence emissions. The assembly of TPE-DPY and CB[ 7 ] presents spherical nanoparticles, exhibiting an emerging phosphorescence emission at 525 nm via the macrocyclic confinement effect to phenyl-pyridine units. CB[ 8 ] with a larger hydrophobic cavity binds with TPE-DPY to form an n:n pseudorotaxane nanorod, which induces an efficient phosphorescence at 545 nm. Varying from the binary assembly of CB[ 7 ] or CB[ 8 ], an entirely distinct topological organic three-dimensional nanoplate is obtained by the co-assembly TPE-DPY with CB[ 7 ]/CB[ 8 ], accompanying enhanced phosphorescence at 540 nm. Uncommonly, the secondary assembly of HACD and TPE-DPY/CB[ 7 ]/CB[ 8 ] activates a single intramolecular PRET process derived from phenyl pyridines unit to methoxy-tetraphenylethylene function group, enabling an NIR delayed fluorescence at 700 nm excited by 333 nm, which ultimately applied to mitochondrial targeted imaging for cancer cells. Physical sciences/Chemistry/Supramolecular chemistry/Self-assembly Physical sciences/Chemistry/Supramolecular chemistry Room-temperature phosphorescence phosphorescence resonance energy transfer large Stokes shift near-infrared emission cell imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Supramolecular assembly based on multiple hydrogen bonds, 1 halogen bonds, 2 metal coordination interactions, 3 and macrocycle encapsulation interactions 4,5 have long been a hot topic in molecular recognition, 6 catalysis, 7–9 luminous materials, 10–13 medicine 14–17 and sensing. 18–19 Among them, the macrocyclic supramolecular assembly has garnered considerable attention due to its potent ability to suppress singlet or triplet exciton vibration, which induces and improves the guest photophysical characteristics, 20–22 especially the room-temperature phosphorescence (RTP) behavior. 23–25 For example, Tian and coworkers reported a series of assembly-induced efficient amorphous RTP materials via modifying phosphor moieties onto β-cyclodextrin (β-CD). 26 Our group constructed a cascade-assembly-enhanced phosphorescence behavior based on CB[ 8 ] and amphiphilic calixarene for cell imaging. 27 Despite the rapid development of RTP systems in recent years, achieving tunable phosphorescence emission, especially in the near-infrared (NIR) region still faces great challenges owing to the limitation of the energy-gap law. 28 Notably, phosphorescence resonance energy transfer (PRET) that transfers T 1 excitons of ultralong organic RTP emitters donor to S 1 excitons of fluorescent chromophores has been proved to be an efficient path for constructing a long-wavelength and long-lifetime delayed fluorescence to achieve tunable afterglow emission, which expands the wide application of RTP materials in bioimaging, 29 sensing, 30 and information anti-counterfeiting. 31,32 George et al. first proposed delayed sensitization of dye singlet states by the phosphorescence resonance energy transfer (PRET) of organic phosphor donors to achieve red afterglow fluorescence. 33 Chi and coworkers described a stepwise PRET system utilizing triphenylene-dyes (Nile red, Cyanine 7)-doped polymers, which earned multicolor afterglow anti-counterfeiting. 34 Li et al. reported an intraparticle-PRET-based near-infrared (NIR) nanoprobe with the aid of amphiphilic triblock copolymers intended for in vivo afterglow imaging. 35 Besides, supramolecular cascade assembly based on macrocyclic confinement-induced phosphorescence by virtue of noncovalent interaction has been proven to be a potential and convenient strategy for constructing PRET in the aqueous phase, enabling not only efficient light-harvesting systems but also NIR-delayed fluorescence for biosensing. 36,37 However, the most reported PRET systems at present are achieved through doping commercial fluorescent dyes or assembly components as acceptors, 38 single intramolecular PRET based on macrocyclic confined guest molecule has been rarely reported to the best of our knowledge. Herein, an efficient single-molecule PRET system based on macrocyclic confinement and polysaccharide mediation was constructed by alkyl-bridged methoxy-tetraphenylethylene-bromophenylpyridines derivative (TPE-DPY), cucurbit[n]uril(n = 7,8), and β-cyclodextrin modified hyaluronic acid (HACD), contributing to the targeted cancer cell imaging with a large Stokes shift of 367 nm and NIR emission. Due to the restriction of phenyl-pyridine unit motion by cucurbituril hydrophobic cavities through host-guest complexation, the binary assembly of CB[ 7 ] or CB[ 8 ] to TPE-DPY all induced a distinct intense phosphorescent emission around 530 nm. By adjusting the ratios of CB[ 7 ] and CB[ 8 ], the supramolecular co-assembly TPE-DPY/CB[ 7 ]/CB[ 8 ] exhibited a stepwise enhanced phosphorescence with phosphorescent lifetime extended from 8.29 µs up to 59.36 µs and presented hierarchical self-assembled three-dimensional nanoplates differing from spherical nanoparticles of TPE-DPY/CB[ 7 ] and pseudorotaxane nanorods of TPE-DPY/CB[ 8 ]. After the further assembly with negatively charged HACD, a single-molecular PRET process derived from phenyl pyridines unit to methoxy-tetraphenylethylene portion was achieved, which was attributed to the change in topological morphology caused by the confinement effect of β-CD to methoxy-tetraphenylethylene group and electrostatic interaction with HA, ultimately giving an NIR delayed fluorescence at 700 nm with a lifetime recorded as 21.6 µs. Taking advantage of the targeting properties of HACD, the aggregate with a large Stokes shift and long-lived NIR photoluminescence was successfully employed for targeted imaging of cancer cells. Results Two kinds of guest molecules, methoxy-tetraphenylethylene derivatives with one (TPE-PY) or two (TPE-DPY) flexible alkyl-bridged phenyl pyridines groups were synthesized by Mizoroki-Heck reaction and alkyl substitution reaction, which were characterized through nuclear magnetic resonance ( 1 H NMR, 13 C NMR) and high-resolution mass spectrometry (HRMS) (Supplementary Figs. 2–10). A series of reference molecules, including alkyl-chain-modified bromophenylpyridinium salts (PY-1), tetraphenylethene derivative possessing vinyl pyridine salts (TPE-1, TPE-2) (Supplementary Fig. 1 and Supplementary Figs. 11–13) were synthesized to process the relevant control experiments for exploring the binding mode of guest molecules with CB[n] (n = 7/8) and the single-molecule PRET luminescence behavior. In contrast to the previously reported mono-bromophenylpyridine derivatives, 39,40 the guest molecule TPE-DPY has two alkyl-bridged bipyridine salt units, which provide more binding sites to assemble with CB[n] (n = 7/8) through ionic dipole interaction and hydrophobic interaction. First, 1 H NMR experiments (Supplementary Fig. 14) and 2D correlation spectroscopy (COSY) (Supplementary Fig. 15) were conducted to investigate the binding behavior between TPE-DPY and CB[ 8 ]. In Supplementary Fig. 14, upon the addition of CB[ 8 ] into the guest solution, the proton signal of TPE-DPY gradually passivated and no longer changed when the CB[ 8 ] concentration exceeded 1 equivalent, indicating the complexation of TPE-DPY and CB[ 8 ] reached an equilibrium stage. Similarly, the UV titration spectrum of TPE-DPY exhibited a persistent red-shift until stabilizing around 1.0 equivalent CB[ 8 ], and the related binding constant was obtained as 5.50 × 10 6 M − 1 (Supplementary Fig. 16). Job’s plot measured by UV-vis spectra confirmed a 1:1 stoichiometric ratio of TPE-DPY to CB[ 8 ] (Supplementary Fig. 17). Furthermore, two-dimensional rotating frame overhauser effect spectroscopy (ROESY) (Supplementary Fig. 18) and two-dimensional diffusion-ordered spectroscopy (DOSY) (Supplementary Fig. 19) were carried out to infer the binding mode. The correlation signals of proton H b’ and H g’ in TPE-DPY (Supplementary Fig. 18) manifested a deep encapsulation of bromophenylpyridine units by CB[ 8 ] cavity in a head-to-tail binding mode. The diffusion coefficients of guest molecule TPE-DPY (D = 2.09×10 − 10 m/s 2 ) and assembly TPE-DPY/CB[ 8 ] (D = 5.19×10 − 11 m/s 2 ) differed by an order of magnitude (Supplementary Fig. 19), which verified the formation of n:n head-to-tail chain supramolecular pseudorotaxane for TPE-DPY/CB[ 8 ]. 41 Correspondingly, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) experiments revealed that the free guest molecule TPE-DPY presented ellipsoidal-shaped nanoparticles with sizes ranging from 50–90 nm (Figs. 3 a, 3 e, 3 i), owing to the hydrophobic interaction and the stacking of tetraphenylethlene groups. In comparison, the TPE-DPY/CB[ 8 ] complex formed a nanorod with a length of approximately 500 nm (Figs. 3 b, 3 f, 3 j), consistent with the head-to-tail chain pseudorotaxane assembly mode. Unlike the complexation of TPE-DPY and CB[ 8 ], Job’s plot determined by UV-vis spectra of TPE-DPY and CB[ 7 ] showed the inflection point at 0.2, implying a 1:4 stoichiometry ratio for TPE-DPY/CB[ 7 ] (Supplementary Fig. 20a). Nevertheless, no meaningful information was captured in 1 H NMR titration experiments of TPE-DPY and CB[ 7 ] because of the strong proton peak passivation following the addition of CB[ 7 ] (Supplementary Fig. 21). Therefore, TPE-PY, consisting of the same functional groups as TPE-DPY, was selected as a reference compound for controlled experiments. Combined the 1 H NMR titration spectrum (Fig. 2 a and Supplementary Fig. 22) and 2D COSY (Supplementary Figs. 23–24), we found that upon the increasing amount of CB[ 7 ] from 0–1 equivalent, an apparent high-field shift of H a and H b on the bromophenylpyridine group was observed resulting from the shielding effect, while the protons H c , H d and H e shifted download indicating that ethylene pyridine moiety was located outside of CB[ 7 ]. As the addition of CB[ 7 ] excessed 1 equivalent, H a and H b underwent a slight shift towards the low field concurrently accompanied by an up-field shift of H c and H d . It indicates that CB[ 7 ] preferentially binds to the bromophenylpyridine section at a low concentration and then assembles with the ethylene pyridine unit at a high concentration. The binding constants obtained through UV titration experiments give more proof for the above results, in which the binding constants of PY-1/CB[ 7 ] were brought to be 9.17 × 10 6 M − 1 higher than 2.72 × 10 5 M − 1 of TPE-2/CB[ 7 ] (Fig. 2 b). Moreover, Job’s plot demonstrated a 1:2 stoichiometric ratio of TPE-PY to CB[ 7 ] (Supplementary Fig. 20b), further confirming the 1:4 binding mode between TPE-DPY and CB[ 7 ]. The assembly of TPE-DPY/CB[ 7 ] was visible in the TEM and SEM images as nanospheres with the increased particle size caused by the rising CB[ 7 ] concentration. As shown in Fig. 3 and Supplementary Fig. 29, a nanosphere measuring roughly 100 nm in diameter was produced by adding 2 equivalents of CB[ 7 ] and a large size nanosphere of about 200 nm developed as the amount of CB[ 7 ] increased to 4 equivalents, owing to the binding effect of CB[ 7 ] for ethylene pyridine portions and bromophenylpyridine groups, which increased the rigidity of supramolecular assembly and the space for stacking arrangement (Figs. 3 c, 3 g, 3 k). Interestingly, entirely varying from the binary assembly of TPE-DPY/CB[ 7 ] and TPE-DPY/CB[ 8 ], the morphology of TPE-DPY/CB[ 7 ]/CB[ 8 ] co-assembly exhibited a polyhedral nanoplate with distinct edges and corners in TEM images (Figs. 3 d and 3 h). Under a scanning electron microscope, it was evident that the three-dimensional nanoplates were formed by hierarchical self-assembly (Fig. 3 l and Supplementary Fig. 29c), which resulted from the side-by-side and layer-by-layer stacking of TPE-DPY/CB[ 7 ]/CB[ 8 ] supramolecular assemblies with a sizeable rigid core and soft chains. The analysis of the co-assembly of the reference molecule TPE-PY with CB[ 7 ] and CB[ 8 ] allowed us to infer the binding mode of the ternary assembly TPE-DPY/CB[ 7 ]/CB[ 8 ]. Specifically, 1 H NMR titration experiment showed that on the basis of TPE-PY/CB[ 7 ] with a stoichiometric ratio of 1:1 where CB[ 7 ] bound to the phenylpyridine unit, vinylpyridine protons (H c , H d , and H e ) exerted an up-field shift upon increase CB[ 8 ] concentration from 0 to 0.5 equivalent, indicating the tight encapsulation of ethylene pyridine moiety by CB[ 7 ] that moved from the phenylpyridine unit (Fig. 2 a and Supplementary Figs. 25–26). Moreover, in the 2D NOESY spectrum of the TPE-PY/CB[ 7 ]/CB[ 8 ] assembly (Supplementary Fig. 27), we could easily locate the cross-peaks between the protons of vinyl functional groups and CB[ 7 ]. The 2:1 stoichiometry ratio obtained from Job’s plot (Supplementary Fig. 28) and the strong binding constant of 2.90×10 12 M − 2 for TPE-PY/CB[ 8 ] (Fig. 2 b) provided further support for the aforementioned findings. These findings suggested that the vinylpyridine and phenyl pyridines moieties of TPE-PY were included in the cavities of CB[ 7 ] and CB[ 8 ], respectively, ultimately generating a ternary supramolecular assembly with a stoichiometric ratio of TPE- PY:CB[ 7 ]:CB[ 8 ] = 2:2:1. Thus, we deduced that the co-assembly of TPE-DPY/CB[ 7 ]/CB[ 8 ] went through a similar assembly process to form a linear supramolecular aggregate with a stoichiometric ratio of 1:2:1. From the above experimental results, it can be seen that CB[n] (n = 7, 8) possesses a different binding affinity to TPE-DPY, leading to a diverse topological morphology for the supramolecular assembly. Profited by the host-guest complexation, hydrophobic interaction, and π – π stacking interactions, the binary assembly of TPE-DPY/CB[ 7 ] presents spherical nanoparticles with adjustable dimensions, and CB[ 8 ] with a larger hydrophobic cavity binds with TPE-DPY to form a n:n rod-shaped pseudorotaxane. The ternary co-assembly TPE-DPY/CB[ 7 ]/CB[ 8 ] has a more robust rigid structure than TPE-DPY/CB[ 7 ] and TPE-DPY/CB[ 8 ], resulting in a linear self-assembly stacked multi-layered three-dimensional nanoplates. Subsequently, the configuration-confined photophysical properties of the assembly of TPE-DPY and CB[n] (n = 7/8) were explored. With the binary assembly of CB[ 7 ] or CB[ 8 ], the absorption peak of TPE-DPY redshifted from 315 nm to 320 nm and 330 nm, respectively, and a comparable red shift by 13 nm occurred in the co-assembly of CB[ 7 ] and CB[ 8 ] (Fig. 2 c). For the photoluminescence spectra shown in Figs. 3 a and 3 b, the guest molecule TPE-DPY exhibited a fluorescence emission at 390 nm with an excitation of 315 nm, and no phosphorescent signal was captured in the delayed spectrum. The assembly of TPE-DPY/2CB[ 7 ], TPE-DPY/4CB[ 7 ], TPE-DPY/CB[ 8 ], and TPE-DPY/CB[ 7 ]/CB[ 8 ] displayed a remarkable new emission peak around 530 nm as compared to the free TPE-DPY (Fig. 4 a). Differing from the steady-state PL spectrum spectra, the delay spectra of these assemblies showed a major emission peak near 530 nm, illustrating its long-lived feature, which was further proved by their microsecond lifetime obtained by the time decay curve measurement (Figs. 4 b, c). Notably, in contrast to the binary assembly TPE-DPY/2CB[ 7 ], TPE-DPY/4CB[ 7 ], and TPE-DPY/CB[ 8 ], the ternary assembly TPE-DPY/CB[ 7 ]/CB[ 8 ] had a stronger luminous intensity with extending the lifetime from 8.29 µs to 59.36 µs (Fig. 4 c), because of the synergistic confinement effect of CB[ 7 ] and CB[ 8 ] on guest molecules and the supramolecular nanostructure formed by the linear rigid assembly layer by layer enabling valid shielding effect on the quencher. Additionally, after the injection of Ar, the lifetime of TPE-DPY/CB[ 7 ]/CB[ 8 ] aqueous solution at 540 nm was significantly increased from 59.36 µs to 129.97 µs (Supplementary Fig. 30) due to the avoidance of the triplet electron quenching caused by oxygen, further confirming the phosphorescence properties of emission peak at 540 nm. The above experiment results demonstrated that the macrocyclic confinement can effectively induce a phosphorescence emission, and the topological morphology of supramolecule assembly can be regulated by adjusting the ratios of CB[ 7 ] and CB[ 8 ], presenting different photophysical properties. On the basis of the binary supramolecular assembly, the multivalent cascade assembly has evolved into an effective method to improve phosphorescence performance. 42 Herein, HACD as a polysaccharide targeting agent has been introduced into TPE-DPY/CB[ 7 ]/CB[ 8 ] to construct a secondary assembly, which resulted in an exchanged topology from hierarchical self-assembled nanoplates to spherical nanoparticles (Figs. 3 d and 5 a). Dynamic light scattering (DLS), TEM, and zeta potential experiments were carried out to explore the assembly behavior for TPE-DPY/CB[ 7 ]/CB[ 8 ]@HACD. DLS measurements suggested that TPE-DPY/CB[ 7 ]/CB[ 8 ]@HACD assembly had an average hydrodynamic diameter of 236 nm, which matched the size of nanospheres in the TEM image (Figs. 5 a, b). Moreover, on the contrary of TPE-DPY and TPE-DPY/CB[ 7 ]/CB[ 8 ] that possessed a positive zeta potential at + 1.74 and + 1.80 mV, respectively, a negative potential value of TPE-DPY/CB[ 7 ]/CB[ 8 ]@HACD was obtained as -0.335 mV (Supplementary Fig. 31), revealing the successful construction of the multicomponent assembly. Remarkably, the cascade assembly of TPE-DPY/CB[ 7 ]/CB[ 8 ]@HACD not only changed the topological morphology but also achieved a HACD-activated single-molecular PRET based on macrocyclic confinement. As shown in Figs. 5 e, 5 f and Supplementary Fig. 32, upon the addition of HACD, the assembly of TPE-DPY/CB[ 7 ]/CB[ 8 ] excited by 333 nm showed a weak phosphorescence at 535 nm with a lifetime of 69.83 µs, and a dominant emission band centered at 700 nm with a lifetime of 21.60 µs, ascribing to the delayed fluorescence of the methoxytetraphenyl-vinylpyridine part stimulated by PRET. To further confirm the multivalent cascade confined single-molecular PRET luminescence behavior, a series of control experiments have been performed. The reference molecule PY-1 displayed CB[ 8 ]-induced strong RTP emission around 520 nm with a lifetime of 388.20 µs (Supplementary Fig. 33), suggesting that the phosphorescence emission of the TPE-DPY assembly emanated from the phenylpyridine units. The steady-state PL spectrum of TPE-1/CB[ 7 ] excited by 450 nm presented an emission peak at 720 nm (Fig. 5 d) with a nanosecond lifetime measured as 2.74 ns, revealing the pure fluorescence properties of methoxy tetraphenylvinylpyridine unit (Supplementary Fig. 34). Similarly, under the excitation of 450 nm, TPE-DPY, TPE-DPY/CB[ 7 ]/CB[ 8 ] and TPE-DPY/CB[ 7 ]/CB[ 8 ]@HACD showed a fluorescence emission of 720 nm, where the lifetime was measured as 0.71 ns, 0.74 ns and 0.85 ns, respectively. No emission signal was obtained in the delay spectrum, verifying the property of HACD-mediated NIR delayed fluorescence at 700 nm that was excited by 333 nm (Supplementary Fig. 35). Furthermore, a large overlap was captured between the absorption spectra of TPE-1/CB[ 7 ] and the phosphorescence spectra of PY-1/CB[ 8 ] (Fig. 5 d), which provided a prerequisite for the PRET derived from phenyl pyridines unit to methoxy-tetraphenylethylene portion within a single-molecule. It is worth noting that no effective PRET phenomenon was obtained in the HACD-assembly doping system with PY-1/CB[ 8 ] as the donor and TPE-1/CB[ 7 ] as the acceptor, which may be caused by the uncontrollable large distance between the donor and the acceptor without covalent connection, hindering the generation of the energy transfer process (Supplementary Fig. 36). Moreover, the addition of bare HA into the TPE-DPY/CB[ 7 ]/CB[ 8 ] solution cannot activate an efficient PRET like HACD, highlighting the important role of β-CD in this cascade assembly process (Supplementary Fig. 37). Accordingly, the binding behavior between the reference TPE-1 and β-CD was investigated by 1 H NMR spectra. It was shown that upon the addition of β-CD, the protons on methoxyphenyl (H 1 , H 2 ) in TPE-1 shifted slightly to low-field, while the protons in styryl pyridiniums remained unchanged (Supplementary Fig. S38), indicating the complexation of β-CD and methoxyphenyl unit. and the association constant of TPE-1/β-CD was determined to be 344.09 M − 1 (Supplementary Fig. 39). These experimental results consistently indicated that the anion effect of HA and the encapsulation of β-CD to methoxy-tetraphenylethylene moiety contributed to the reconstruction of topological morphology for TPE-DPY/CB[ 7 ]/CB[ 8 ], which facilitated the single intramolecular PRET process leading to 700 nm NIR delayed fluorescence emission with large Stokes shift of 367 nm (Fig. 5 c). Commonly, the phosphorescence spectra of the assemblies TPE-DPY/CB[ 7 ]@HACD and TPE-DPY/CB[ 8 ]@HACD also exhibited NIR emission peaks at 700 nm, implying the universality of HACD activation for single-molecular PRET process (Supplementary Fig. 40). In order to explore the application of macrocyclic confinement and HACD-activated single-molecule PRET system, cell imaging experiments were constructed. First, Human cervical carcinoma cells (Hela cells) and human embryonic kidney cells (293T cells) were treated with TPE-DPY/CB[ 7 ]/CB[ 8 ]@HACD for 12h, respectively, and then incubated with Hoechst and Mito-Tracker Green for localization experiment. The confocal laser scanning microscopy (CLSM) experiments were performed to investigate the intracellular NIR emission signals. As shown in Figs. 6 a and 6 c, Hela cells exhibited a bright NIR luminescence in a red channel (650–750 nm), whereas almost no red emission signal was found for normal 293T cells. These imaging results implied that TPE-DPY/CB[ 7 ]/CB[ 8 ]@HACD was preferentially internalized by cancer cells rather than normal cells, which may be caused by the overexpressed HA receptors for cancer cells. Furthermore, colocalization analysis demonstrated that the NIR luminescence signal overlapped well with the green signal of Mito Tracker, which corresponded to the yellow region in the merged image (Fig. 6 b). It revealed the ability of TPE-DPY/CB[ 7 ]/CB[ 8 ]@HACD for targeted mitochondria imaging in cancer cells, and the high Pearson correlation provided strong evidence for this result (Supplementary Fig. 41). Finally, CCK-8 assays were conducted to evaluate cytotoxicity experiments on the above two cells, and the high survival rate indicated low cytotoxicity of the assembly TPE-DPY/CB[ 7 ]/CB[ 8 ]@HACD (Supplementary Fig. 42). Discussion In summary, a single-molecule PRET is activated by the macrocyclic confinement of CB[n] (n = 7,8) and the secondary assembly of HACD to achieve an NIR-delayed fluorescence emission. Based on the different cavity sizes, the primary assembly of CB[ 7 ] and CB[ 8 ] to TPE-DPY presented a macrocyclic confinement-induced phosphorescence behavior accompanied by controllable topological morphologies, which realized a transformation from nanosphere, rod-shaped pseudorotaxane to hierarchical self-assembled nanoplate. Especially, the secondary assembly of HACD activated the single intramolecular PRET from the phenyl pyridines unit to the methoxy-tetraphenylethylene portion, generating an NIR delayed fluorescence emission at 700 nm. Different from the dye-doped PRET system, this macrocyclic-confinement polysaccharide activated single molecular PRET system displayed a large Stokes shift of 367 nm and was successfully applied in mitochondrial-targeted imaging for cancer cells, which provides a new approach for the construction and application of single-molecule PRET. Methods Materials. Except for additional stated, all reagents and solvents were available from commercial sources and used directly without any purification. HA-CD that we used was synthesized on the basis of the literature, indicating the degree of substitution (DS) was 16.43%. 1 H NMR and 13 C NMR spectrums were recorded through Bruker AV400. Two-dimensional NMR (COSY, DOSY, NOESY, and ROESY) spectra were measured on Bruker AVANCE III HD 400 spectrometer. High-resolution mass spectrometry (HR-MS) was recorded on a Q-TOF LC-MS in an Electrospray ionization (ESI) source. UV-vis absorption was kept details on Shimadzu UV-3600 spectrophotometer with a PTC-348WI temperature controller at 298 K. Photoluminescence (PL) spectrum and time-correlated decay profiles were documented on Edinburgh Instruments FS5 (Livingstone, UK). The TEM experiment was carried out on FEI Tecnai G2 F20 under 200 KV. SEM was accomplished on FEI Apreo S LoVac scanning electronic microscope working at an accelerating voltage of 30 keV. The Zeta potentials were examined on Brookhaven ZETAPALS/BI-200SM at 298 K. Dynamic Light Scattering (DLS) was determined by using a laser lights-scattering spectrometer (BI-200SM) equipped with a digital correlator (Turbo Corr) at 635 nm at a scattering angle of 90°. Cell images were captured on Olympus FV1000 Laser scanning confocal microscope. Cytotoxicity experiments. The cells, including Hela cells and 293T cells, were all gained from the Cell Resource Center of China Academy of Medical Science in Beijing. These cells were cultured in particular conditions with the addition of 10% FBS and 1% penicillin/streptomycin in the DMEM nutrient medium and humidified incubator with 5% CO 2 atmosphere at 37 ℃. The Hela cells and 293T cells were incubated with TPE-DPY/CB[ 7 ]/CB[ 8 ]@HACD at distinct concentrations in 96-well plates for 24 hours. The relative cellular viability was determined by the CCK8 assay. Cell imaging experiments. The Hela cells and 293T cells were precultured in confocal petri dishes, respectively, for 24 h. Then, the well-cultured cells were incubated with TPE-DPY/CB[ 7 ]/CB[ 8 ]@HACD (20 µM) for a further 2 h. The cells were stained with Hoechst (1 nM) and Mito-Tracker Green (1 nM) for half an hour, washed three times with PBS, and then added PBS (1 ml) to observe by microscope. Synthesis of compound TPE-DPY. Under N 2 protection, 4,4′-[[2,2-bis(4-methoxyphenyl)ethenylidene]bis(4,1-phenylene-2,1-ethenediyl)]pyridine (54 mg, 0.09 mmol) and PY-1 (94.5 mg, 0.22 mmol) were dissolved in CH 3 CN (5 ml). The reaction mixture was heated to 85 ℃ for 36 hours. After the reaction, CH 3 CN was evaporated, and acetone was used for ultrasound cleaning. Then, the mixture was filtered and washed with acetone twice. Additionally, the crude powder was purified by heat filtration and recrystallization to give an orange solid (18 mg, yield: 13.6%). 1 H NMR (400 MHz, Methanol- d 4 ) δ 9.07 (d, J = 6.7 Hz, 4H), 8.86 (d, J = 6.5 Hz, 4H), 8.49 (d, J = 6.5 Hz, 4H), 8.20 (d, J = 6.6 Hz, 4H), 7.97 (d, J = 8.5 Hz, 4H), 7.91 (d, J = 16.2 Hz, 2H), 7.84 (d, J = 8.5 Hz, 4H), 7.56 (d, J = 8.1 Hz, 4H), 7.39 (d, J = 16.2 Hz, 2H), 7.15 (d, J = 8.2 Hz, 4H), 6.98 (d, J = 8.6 Hz, 4H), 6.72 (d, J = 8.7 Hz, 4H), 4.84 (s, 4H), 4.76 (t, J = 7.5 Hz, 4H), 3.75 (s, 6H), 2.84–2.78 (m, 4H).; 13 C NMR (101 MHz, Methanol- d 4 ) δ 158.97, 155.76, 154.58, 146.95, 144.83, 143.98, 141.73, 135.52, 133.19, 132.80, 132.42, 131.95, 129.61, 127.80, 127.00, 124.92, 124.01, 122.16, 112.94, 57.27, 56.79, 54.26, 31.99.; HRMS (ESI) m/z for C 70 H 62 Br 6 N 4 O 2 calcd. [M-4Br] 4+ 287.5799, found: 287.0806. Declarations Data Availability Data supporting the findings of this study are included within the article and Supplementary Information files. Data are available from the authors upon request. Competing interests The authors declare no competing interests. Author contributions X.Z. and X.B. contributed equally to this work and were primarily responsible for the experiments, then measured and analyzed the experiment data. X.Z. wrote the manuscript. H-Y Z. and L-H W. gave valuable suggestions. Y.L. supervised the work and edited the manuscript. All authors analyzed and discussed the results and reviewed the manuscript. Acknowledgements We thank National Nature Science Foundation of China (NNSFC, Grant Nos. 22131008 and 22271165, China Fundamental Research Funds for the Central Universities and the Haihe Laboratory of Sustainable Chemical Transformations for financial support. References Prins LJ, Reinhoudt DN, Timmerman P (2001) Noncovalent synthesis using hydrogen bonding. 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Nat Commun 13:4185 Huang Z, Song W, Chen X (2020) Supramolecular Self-Assembled Nanostructures for Cancer Immunotherapy. Front Chem 8:380 Zhao L, Liu Y, Xing R, Yan X (2020) Supramolecular Photothermal Effects: A Promising Mechanism for Efficient Thermal Conversion. Angew Chem Int Ed 59:3793–3801 Zhang SG (2003) Fabrication of novel biomaterials through molecular self-assembly. Nat Biotechnol 21:1171–1178 Zhang J, Ma PX (2013) Cyclodextrin-based supramolecular systems for drug delivery: Recent progress and future perspective. Adv Drug Deliv Rev 65:1215–1233 Li B et al (2022) Supramolecular Assembly of Organoplatinum(II) Complexes for Subcellular Distribution and Cell Viability Monitoring with Differentiated Imaging. Angew Chem Int Ed 61:e202210703 Dai D, Yang J, Yang Y-W (2022) Supramolecular Assemblies with Aggregation-Induced Emission Properties for Sensing and Detection. 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Angew Chem Int Ed 61:e202115748 Gui H, Huang Z, Yuan Z, Ma X (2022) Ambient White-Light Afterglow Emission Based on Triplet-to-Singlet Förster Resonance Energy Transfer. CCS Chem 4:173–181 Li D et al (2022) Completely aqueous processable stimulus responsive organic room temperature phosphorescence materials with tunable afterglow color. Nat Commun 13:347 Kuila S, George SJ (2020) Phosphorescence Energy Transfer: Ambient Afterglow Fluorescence from Water-Processable and Purely Organic Dyes via Delayed Sensitization. Angew Chem Int Ed 59:9393–9397 Lin F et al (2022) Stepwise Energy Transfer: Near-Infrared Persistent Luminescence from Doped Polymeric Systems. Adv Mater 34:2108333 Dang Q et al (2020) Room-Temperature Phosphorescence Resonance Energy Transfer for Construction of Near-Infrared Afterglow Imaging Agents. Adv Mater 32:2006752 Liu Z, Lin W, Liu Y (2022) Macrocyclic Supramolecular Assemblies Based on Hyaluronic Acid and Their Biological Applications. Acc Chem Res 55:3417–3429 Dai X-Y, Huo M, Dong X, Hu Y-Y, Liu Y (2022) Noncovalent Polymerization-Activated Ultrastrong Near-Infrared Room-Temperature Phosphorescence Energy Transfer Assembly in Aqueous Solution. Adv Mater 34:2203534 Dai X-Y, Huo M, Liu Y (2023) Phosphorescence resonance energy transfer from purely organic supramolecular assembly. Nat Rev Chem 7:854–874 Ma X-K et al (2021) A twin-axial pseudorotaxane for phosphorescence cell imaging. Chem Commun 57:1214–1217 Zhang Z-Y et al (2020) A Synergistic Enhancement Strategy for Realizing Ultralong and Efficient Room-Temperature Phosphorescence. Angew Chem Int Ed 59:18748–18754 Yu H-J et al (2021) Photooxidation-Driven Purely Organic Room-Temperature Phosphorescent Lysosome-Targeted Imaging. J Am Chem Soc 143:13887–13894 Huo M, Dai X-Y, Liu Y (2021) Ultrahigh Supramolecular Cascaded Room-Temperature Phosphorescence Capturing System. Angew Chem Int Ed 60:27171–27177 Additional Declarations There is NO Competing Interest. Supplementary Files Supportinginformation.docx Cite Share Download PDF Status: Published Journal Publication published 05 Jun, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3784778","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":262073976,"identity":"8ad1663b-4283-469a-bfab-896a58b1fd4c","order_by":0,"name":"Yu Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYFAC5oYDDBVAmoeB4QCQR4wWRqCWM6RqYWBsg2hhIEqLwY3ExsO88+7Im/Mcfgh0oXViA/vZA4S0NBzm3fbMcGdvmwHQhemJDTx5CcRoOcy44TyDwQHGtsOJDRI8BkRomXPYfsN59g8HGP8RraXhcOKGsz1AWxqI0CJ55mHDwTnHDidvOHOm4EDCsXTjNp4c/Fr4jicf/vCm5rDthjPpmz98qLGW7Wc/g1+LwgFkXgIQs+FVDwTyDYRUjIJRMApGwSgAANUfUk0V9+S4AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-8723-1896","institution":"Nankai University","correspondingAuthor":true,"prefix":"","firstName":"Yu","middleName":"","lastName":"Liu","suffix":""},{"id":262073977,"identity":"92478095-f05f-47ab-88e7-bfb7c1bed755","order_by":1,"name":"Xiaolu Zhou","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Xiaolu","middleName":"","lastName":"Zhou","suffix":""},{"id":262073978,"identity":"cf0b3578-b2e8-40e9-87eb-c3723cc007c4","order_by":2,"name":"Xue Bai","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Xue","middleName":"","lastName":"Bai","suffix":""},{"id":262073979,"identity":"28f566c7-423f-4217-9aa6-3022593dde6d","order_by":3,"name":"Heng-Yi Zhang","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Heng-Yi","middleName":"","lastName":"Zhang","suffix":""},{"id":262073980,"identity":"67978d01-f635-4a78-a94c-d230a6b21e30","order_by":4,"name":"Li-Hua Wang","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Li-Hua","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2023-12-21 04:35:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3784778/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3784778/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-024-49238-5","type":"published","date":"2024-06-05T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49326936,"identity":"b135eaf1-2c74-4779-b66a-bd76dbae778e","added_by":"auto","created_at":"2024-01-08 17:34:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2160231,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of the tunable self-assembly mechanism between TPE-DPY, CB[7] and CB[8], as well as the single-molecular PRET process in assembly.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3784778/v1/fe1840c326da3a7186433df9.png"},{"id":49326939,"identity":"e781055e-c46d-4ab7-b0d9-81c2a51b570f","added_by":"auto","created_at":"2024-01-08 17:34:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1803999,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of binding behavior between TPE-PY and CB[7]/CB[8]. (a) \u003c/strong\u003e\u003csup\u003e1\u003c/sup\u003eH NMR spectra (400 MHz, D\u003csub\u003e2\u003c/sub\u003eO with 10% DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, 298 K) of TPE-PY (red), TPE-PY:CB[7] = 1:1 (black), TPE-PY:CB[7] = 1:2 (green), TPE-PY:CB[7]:CB[8] = 2:2:1 (blue); \u003cstrong\u003e(b)\u003c/strong\u003e From left to right, the binding constants of PY-1 and TPE-2 with the addition of CB[7], and TPE-PY with the addition of CB[8]. \u003cstrong\u003e(c)\u003c/strong\u003e UV-vis absorption of TPE-DPY ([TPE-DPY] = 1×10\u003csup\u003e-5\u003c/sup\u003e M), TPE-DPY/2CB[7] ([TPE-DPY] = 1×10\u003csup\u003e-5\u003c/sup\u003e M, [CB[7]] = 2×10\u003csup\u003e-5\u003c/sup\u003e M), TPE-DPY/4CB[7] ([TPE-DPY] = 1×10\u003csup\u003e-5\u003c/sup\u003e M, [CB[7]] = 4×10\u003csup\u003e-5\u003c/sup\u003e M), TPE-DPY/CB[8] ([TPE-DPY] = 1×10\u003csup\u003e-5\u003c/sup\u003e M, [CB[8]] = 1×10\u003csup\u003e-5\u003c/sup\u003e M), TPE-DPY/CB[7]/CB[8]. ([TPE-DPY] = 1×10\u003csup\u003e-5\u003c/sup\u003e M, [CB[7]] = 2×10\u003csup\u003e-5\u003c/sup\u003e M, [CB[8]] = 1×10\u003csup\u003e-5\u003c/sup\u003e M).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3784778/v1/e95e209dcda8093530d0d110.png"},{"id":49326941,"identity":"29011d07-bf82-4043-84ac-fba4ed3bb994","added_by":"auto","created_at":"2024-01-08 17:34:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3978603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTopological morphology characterization of TPE-DPY and the assemblies. (a-d) \u003c/strong\u003e3D models of assembled structures.\u003cstrong\u003e (e-h) \u003c/strong\u003eTEM images of TPE-DPY ([TPE-DPY] = 1×10\u003csup\u003e-5\u003c/sup\u003e M), TPE-DPY/CB[8] ([TPE-DPY] = 1×10\u003csup\u003e-5\u003c/sup\u003e M, [CB[8]] = 1×10\u003csup\u003e-5\u003c/sup\u003e M), TPE-DPY/4CB[7] ([TPE-DPY] = 5×10\u003csup\u003e-6\u003c/sup\u003e M, [CB[7]] = 2×10\u003csup\u003e-5\u003c/sup\u003e M), TPE-DPY/CB[7]/CB[8]. ([TPE-DPY] = 1×10\u003csup\u003e-5\u003c/sup\u003e M, [CB[7]] = 2×10\u003csup\u003e-5\u003c/sup\u003e M, [CB[8]] = 1×10\u003csup\u003e-5\u003c/sup\u003e M) (from left to right). \u003cstrong\u003e(i-l) \u003c/strong\u003eSEM images of TPE-DPY, TPE-DPY/CB[8], TPE-DPY/4CB[7] and TPE-DPY/CB[7]/CB[8].\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3784778/v1/bcbfa804ba45a53c3d05eb95.png"},{"id":49326938,"identity":"3e090e46-d618-440b-a2d3-76eab2a9ae2a","added_by":"auto","created_at":"2024-01-08 17:34:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1019817,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhotophysical properties of TPE-DPY and the assemblies. (a, b)\u003c/strong\u003e The steady-state PL spectra (\u003cstrong\u003ea)\u003c/strong\u003e and phosphorescence spectra (\u003cstrong\u003eb)\u003c/strong\u003e of TPE-DPY ([TPE-DPY] = 1×10\u003csup\u003e-5\u003c/sup\u003e M, λ\u003csub\u003eex\u003c/sub\u003e = 315 nm), TPE-DPY/2CB[7] ([TPE-DPY] = 1×10\u003csup\u003e-5\u003c/sup\u003e M, [CB[7]] = 2×10\u003csup\u003e-5\u003c/sup\u003e M, λ\u003csub\u003eex\u003c/sub\u003e = 318 nm), TPE-DPY/4CB[7] ([TPE-DPY] = 1×10\u003csup\u003e-5\u003c/sup\u003e M, [CB[7]] = 4×10\u003csup\u003e-5\u003c/sup\u003e M, λ\u003csub\u003eex\u003c/sub\u003e = 320 nm), TPE-DPY/CB[8] ([TPE-DPY] = 1×10\u003csup\u003e-5\u003c/sup\u003e M, [CB[8]] = 1×10\u003csup\u003e-5\u003c/sup\u003e M, λ\u003csub\u003eex\u003c/sub\u003e = 330 nm), TPE-DPY/CB[7]/CB[8] ([TPE-DPY] = 1×10\u003csup\u003e-5\u003c/sup\u003e M, [CB[7]] = 2×10\u003csup\u003e-5\u003c/sup\u003e M, [CB[8]] = 1×10\u003csup\u003e-5\u003c/sup\u003e M, λ\u003csub\u003eex\u003c/sub\u003e = 333 nm) (delay = 50 μs). \u003cstrong\u003e(c)\u003c/strong\u003e Time-resolved PL decay curves of TPE-DPY/2CB[7], TPE-DPY/4CB[7], TPE-DPY/CB[8] and TPE-DPY/CB[7]/CB[8] in aqueous solution.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3784778/v1/4fd316fec6df5d88a0f9260f.png"},{"id":49326937,"identity":"c0a0b70d-1b2f-42ab-b572-4cddfdcbe1b9","added_by":"auto","created_at":"2024-01-08 17:34:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2155482,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHACD-activated PRET process for the secondary assembly. (a)\u003c/strong\u003e TEM image of TPE-DPY/CB[7]/CB[8]@HACD. \u003cstrong\u003e(b)\u003c/strong\u003e Size distribution of TPE-DPY/CB[7]/CB[8]@HACD determined by dynamic light scattering. (\u003cstrong\u003ec)\u003c/strong\u003e Schematic illustration of HACD mediated efficient single-molecule PRET process. \u003cstrong\u003e(d)\u003c/strong\u003e Normalized phosphorescence emission spectrum of PY-1/CB[8] ([PY-1] = 2.5×10\u003csup\u003e-5 \u003c/sup\u003eM, [CB[8]] = 1.25×10\u003csup\u003e-5\u003c/sup\u003e M), and the excitation and emission spectra of TPE-1/CB[7] ([TPE-1] = 2.5×10\u003csup\u003e-5 \u003c/sup\u003eM, [CB[7]] = 5×10\u003csup\u003e-5\u003c/sup\u003e M). \u003cstrong\u003e(e)\u003c/strong\u003e Phosphorescence spectra of TPE-DPY/CB[7]/CB[8] upon the addition of 0-0.045 mg/ml HACD (([TPE-DPY] = 2.5×10\u003csup\u003e-5 \u003c/sup\u003eM, [CB[7]] = 5×10\u003csup\u003e-5\u003c/sup\u003e M, [CB[8]] = 2.5×10\u003csup\u003e-5\u003c/sup\u003e M). \u003cstrong\u003e(f)\u003c/strong\u003e The time-resolved PL decay curves of TPE-DPY/CB[7]/CB[8]@HACD aqueous solution record at 530 nm and 700 nm.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3784778/v1/c476cf8e816b72c940b70712.png"},{"id":49326940,"identity":"c935844c-d63e-4907-a760-73cca13c05e2","added_by":"auto","created_at":"2024-01-08 17:34:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1651298,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApplication of TPE-DPY/CB[7]/CB[8]@HACD in targeted imaging for cancer cells. (a,b)\u003c/strong\u003e Confocal microscopy images and merged images of Hela cells in the presence of TPE-DPY/CB[7]/CB[8]@HACD, Hoechst and Mito-Tracker Green. \u003cstrong\u003e(c)\u003c/strong\u003e Confocal microscopy images and merged images of 293T cells in the presence of TPE-DPY/CB[7]/CB[8]@HACD and Hoechst. ([TPE-DPY] = 2×10\u003csup\u003e-5\u003c/sup\u003e M, [CB[7]] = 4×10\u003csup\u003e-5\u003c/sup\u003e M, [CB[8]] = 2×10\u003csup\u003e-5\u003c/sup\u003e M, [HACD] = 0.036 mg/ml, Green channel: 450 nm – 550 nm, red channel: 650 nm – 800 nm).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3784778/v1/2d7c8b2514baba61ecd9b509.png"},{"id":57826153,"identity":"501912c1-c489-49df-bbbf-832fcd9c18ba","added_by":"auto","created_at":"2024-06-06 07:07:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16625061,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3784778/v1/0c308f00-08ab-40fe-bddd-b26b76bf306c.pdf"},{"id":49326962,"identity":"60207fd1-cdb5-4a41-8c77-8867b15b2db7","added_by":"auto","created_at":"2024-01-08 17:34:24","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":34787257,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Supportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-3784778/v1/33bac202bf32ac3df6078f8e.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Single-Molecule Phosphorescence Resonance Energy Transfer for NIR Targeted Cell Imaging","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSupramolecular assembly based on multiple hydrogen bonds,\u003csup\u003e1\u003c/sup\u003e halogen bonds,\u003csup\u003e2\u003c/sup\u003e metal coordination interactions,\u003csup\u003e3\u003c/sup\u003e and macrocycle encapsulation interactions\u003csup\u003e4,5\u003c/sup\u003e have long been a hot topic in molecular recognition,\u003csup\u003e6\u003c/sup\u003e catalysis,\u003csup\u003e7\u0026ndash;9\u003c/sup\u003e luminous materials,\u003csup\u003e10\u0026ndash;13\u003c/sup\u003e medicine\u003csup\u003e14\u0026ndash;17\u003c/sup\u003e and sensing.\u003csup\u003e18\u0026ndash;19\u003c/sup\u003e Among them, the macrocyclic supramolecular assembly has garnered considerable attention due to its potent ability to suppress singlet or triplet exciton vibration, which induces and improves the guest photophysical characteristics,\u003csup\u003e20\u0026ndash;22\u003c/sup\u003e especially the room-temperature phosphorescence (RTP) behavior.\u003csup\u003e23\u0026ndash;25\u003c/sup\u003e For example, Tian and coworkers reported a series of assembly-induced efficient amorphous RTP materials via modifying phosphor moieties onto β-cyclodextrin (β-CD).\u003csup\u003e26\u003c/sup\u003e Our group constructed a cascade-assembly-enhanced phosphorescence behavior based on CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and amphiphilic calixarene for cell imaging.\u003csup\u003e27\u003c/sup\u003e Despite the rapid development of RTP systems in recent years, achieving tunable phosphorescence emission, especially in the near-infrared (NIR) region still faces great challenges owing to the limitation of the energy-gap law.\u003csup\u003e28\u003c/sup\u003e Notably, phosphorescence resonance energy transfer (PRET) that transfers T\u003csub\u003e1\u003c/sub\u003e excitons of ultralong organic RTP emitters donor to S\u003csub\u003e1\u003c/sub\u003e excitons of fluorescent chromophores has been proved to be an efficient path for constructing a long-wavelength and long-lifetime delayed fluorescence to achieve tunable afterglow emission, which expands the wide application of RTP materials in bioimaging,\u003csup\u003e29\u003c/sup\u003e sensing,\u003csup\u003e30\u003c/sup\u003e and information anti-counterfeiting.\u003csup\u003e31,32\u003c/sup\u003e George et al. first proposed delayed sensitization of dye singlet states by the phosphorescence resonance energy transfer (PRET) of organic phosphor donors to achieve red afterglow fluorescence.\u003csup\u003e33\u003c/sup\u003e Chi and coworkers described a stepwise PRET system utilizing triphenylene-dyes (Nile red, Cyanine 7)-doped polymers, which earned multicolor afterglow anti-counterfeiting.\u003csup\u003e34\u003c/sup\u003e Li et al. reported an intraparticle-PRET-based near-infrared (NIR) nanoprobe with the aid of amphiphilic triblock copolymers intended for in vivo afterglow imaging.\u003csup\u003e35\u003c/sup\u003e Besides, supramolecular cascade assembly based on macrocyclic confinement-induced phosphorescence by virtue of noncovalent interaction has been proven to be a potential and convenient strategy for constructing PRET in the aqueous phase, enabling not only efficient light-harvesting systems but also NIR-delayed fluorescence for biosensing.\u003csup\u003e36,37\u003c/sup\u003e However, the most reported PRET systems at present are achieved through doping commercial fluorescent dyes or assembly components as acceptors,\u003csup\u003e38\u003c/sup\u003e single intramolecular PRET based on macrocyclic confined guest molecule has been rarely reported to the best of our knowledge.\u003c/p\u003e \u003cp\u003eHerein, an efficient single-molecule PRET system based on macrocyclic confinement and polysaccharide mediation was constructed by alkyl-bridged methoxy-tetraphenylethylene-bromophenylpyridines derivative (TPE-DPY), cucurbit[n]uril(n\u0026thinsp;=\u0026thinsp;7,8), and β-cyclodextrin modified hyaluronic acid (HACD), contributing to the targeted cancer cell imaging with a large Stokes shift of 367 nm and NIR emission. Due to the restriction of phenyl-pyridine unit motion by cucurbituril hydrophobic cavities through host-guest complexation, the binary assembly of CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] or CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] to TPE-DPY all induced a distinct intense phosphorescent emission around 530 nm. By adjusting the ratios of CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], the supramolecular co-assembly TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] exhibited a stepwise enhanced phosphorescence with phosphorescent lifetime extended from 8.29 \u0026micro;s up to 59.36 \u0026micro;s and presented hierarchical self-assembled three-dimensional nanoplates differing from spherical nanoparticles of TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and pseudorotaxane nanorods of TPE-DPY/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. After the further assembly with negatively charged HACD, a single-molecular PRET process derived from phenyl pyridines unit to methoxy-tetraphenylethylene portion was achieved, which was attributed to the change in topological morphology caused by the confinement effect of β-CD to methoxy-tetraphenylethylene group and electrostatic interaction with HA, ultimately giving an NIR delayed fluorescence at 700 nm with a lifetime recorded as 21.6 \u0026micro;s. Taking advantage of the targeting properties of HACD, the aggregate with a large Stokes shift and long-lived NIR photoluminescence was successfully employed for targeted imaging of cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTwo kinds of guest molecules, methoxy-tetraphenylethylene derivatives with one (TPE-PY) or two (TPE-DPY) flexible alkyl-bridged phenyl pyridines groups were synthesized by Mizoroki-Heck reaction and alkyl substitution reaction, which were characterized through nuclear magnetic resonance (\u003csup\u003e1\u003c/sup\u003eH NMR, \u003csup\u003e13\u003c/sup\u003eC NMR) and high-resolution mass spectrometry (HRMS) (Supplementary Figs.\u0026nbsp;2\u0026ndash;10). A series of reference molecules, including alkyl-chain-modified bromophenylpyridinium salts (PY-1), tetraphenylethene derivative possessing vinyl pyridine salts (TPE-1, TPE-2) (Supplementary Fig.\u0026nbsp;1 and Supplementary Figs.\u0026nbsp;11\u0026ndash;13) were synthesized to process the relevant control experiments for exploring the binding mode of guest molecules with CB[n] (n\u0026thinsp;=\u0026thinsp;7/8) and the single-molecule PRET luminescence behavior. In contrast to the previously reported mono-bromophenylpyridine derivatives,\u003csup\u003e39,40\u003c/sup\u003e the guest molecule TPE-DPY has two alkyl-bridged bipyridine salt units, which provide more binding sites to assemble with CB[n] (n\u0026thinsp;=\u0026thinsp;7/8) through ionic dipole interaction and hydrophobic interaction. First, \u003csup\u003e1\u003c/sup\u003eH NMR experiments (Supplementary Fig.\u0026nbsp;14) and 2D correlation spectroscopy (COSY) (Supplementary Fig.\u0026nbsp;15) were conducted to investigate the binding behavior between TPE-DPY and CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In Supplementary Fig.\u0026nbsp;14, upon the addition of CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] into the guest solution, the proton signal of TPE-DPY gradually passivated and no longer changed when the CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] concentration exceeded 1 equivalent, indicating the complexation of TPE-DPY and CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] reached an equilibrium stage. Similarly, the UV titration spectrum of TPE-DPY exhibited a persistent red-shift until stabilizing around 1.0 equivalent CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and the related binding constant was obtained as 5.50 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;16). Job\u0026rsquo;s plot measured by UV-vis spectra confirmed a 1:1 stoichiometric ratio of TPE-DPY to CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] (Supplementary Fig.\u0026nbsp;17). Furthermore, two-dimensional rotating frame overhauser effect spectroscopy (ROESY) (Supplementary Fig.\u0026nbsp;18) and two-dimensional diffusion-ordered spectroscopy (DOSY) (Supplementary Fig.\u0026nbsp;19) were carried out to infer the binding mode. The correlation signals of proton H\u003csub\u003eb\u0026rsquo;\u003c/sub\u003e and H\u003csub\u003eg\u0026rsquo;\u003c/sub\u003e in TPE-DPY (Supplementary Fig.\u0026nbsp;18) manifested a deep encapsulation of bromophenylpyridine units by CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] cavity in a head-to-tail binding mode. The diffusion coefficients of guest molecule TPE-DPY (D\u0026thinsp;=\u0026thinsp;2.09\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e m/s\u003csup\u003e2\u003c/sup\u003e) and assembly TPE-DPY/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] (D\u0026thinsp;=\u0026thinsp;5.19\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;11\u003c/sup\u003e m/s\u003csup\u003e2\u003c/sup\u003e) differed by an order of magnitude (Supplementary Fig.\u0026nbsp;19), which verified the formation of n:n head-to-tail chain supramolecular pseudorotaxane for TPE-DPY/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003csup\u003e41\u003c/sup\u003e Correspondingly, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) experiments revealed that the free guest molecule TPE-DPY presented ellipsoidal-shaped nanoparticles with sizes ranging from 50\u0026ndash;90 nm (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei), owing to the hydrophobic interaction and the stacking of tetraphenylethlene groups. In comparison, the TPE-DPY/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] complex formed a nanorod with a length of approximately 500 nm (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej), consistent with the head-to-tail chain pseudorotaxane assembly mode.\u003c/p\u003e \u003cp\u003eUnlike the complexation of TPE-DPY and CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], Job\u0026rsquo;s plot determined by UV-vis spectra of TPE-DPY and CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] showed the inflection point at 0.2, implying a 1:4 stoichiometry ratio for TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] (Supplementary Fig.\u0026nbsp;20a). Nevertheless, no meaningful information was captured in \u003csup\u003e1\u003c/sup\u003eH NMR titration experiments of TPE-DPY and CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] because of the strong proton peak passivation following the addition of CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] (Supplementary Fig.\u0026nbsp;21). Therefore, TPE-PY, consisting of the same functional groups as TPE-DPY, was selected as a reference compound for controlled experiments. Combined the \u003csup\u003e1\u003c/sup\u003eH NMR titration spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and Supplementary Fig.\u0026nbsp;22) and 2D COSY (Supplementary Figs.\u0026nbsp;23\u0026ndash;24), we found that upon the increasing amount of CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] from 0\u0026ndash;1 equivalent, an apparent high-field shift of H\u003csub\u003ea\u003c/sub\u003e and H\u003csub\u003eb\u003c/sub\u003e on the bromophenylpyridine group was observed resulting from the shielding effect, while the protons H\u003csub\u003ec\u003c/sub\u003e, H\u003csub\u003ed\u003c/sub\u003e and H\u003csub\u003ee\u003c/sub\u003e shifted download indicating that ethylene pyridine moiety was located outside of CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. As the addition of CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] excessed 1 equivalent, H\u003csub\u003ea\u003c/sub\u003e and H\u003csub\u003eb\u003c/sub\u003e underwent a slight shift towards the low field concurrently accompanied by an up-field shift of H\u003csub\u003ec\u003c/sub\u003e and H\u003csub\u003ed\u003c/sub\u003e. It indicates that CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] preferentially binds to the bromophenylpyridine section at a low concentration and then assembles with the ethylene pyridine unit at a high concentration. The binding constants obtained through UV titration experiments give more proof for the above results, in which the binding constants of PY-1/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] were brought to be 9.17 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e higher than 2.72 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of TPE-2/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Moreover, Job\u0026rsquo;s plot demonstrated a 1:2 stoichiometric ratio of TPE-PY to CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] (Supplementary Fig.\u0026nbsp;20b), further confirming the 1:4 binding mode between TPE-DPY and CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The assembly of TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] was visible in the TEM and SEM images as nanospheres with the increased particle size caused by the rising CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] concentration. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Supplementary Fig.\u0026nbsp;29, a nanosphere measuring roughly 100 nm in diameter was produced by adding 2 equivalents of CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and a large size nanosphere of about 200 nm developed as the amount of CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] increased to 4 equivalents, owing to the binding effect of CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] for ethylene pyridine portions and bromophenylpyridine groups, which increased the rigidity of supramolecular assembly and the space for stacking arrangement (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek).\u003c/p\u003e \u003cp\u003eInterestingly, entirely varying from the binary assembly of TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and TPE-DPY/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], the morphology of TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] co-assembly exhibited a polyhedral nanoplate with distinct edges and corners in TEM images (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). Under a scanning electron microscope, it was evident that the three-dimensional nanoplates were formed by hierarchical self-assembly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el and Supplementary Fig.\u0026nbsp;29c), which resulted from the side-by-side and layer-by-layer stacking of TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] supramolecular assemblies with a sizeable rigid core and soft chains. The analysis of the co-assembly of the reference molecule TPE-PY with CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] allowed us to infer the binding mode of the ternary assembly TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Specifically, \u003csup\u003e1\u003c/sup\u003eH NMR titration experiment showed that on the basis of TPE-PY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] with a stoichiometric ratio of 1:1 where CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] bound to the phenylpyridine unit, vinylpyridine protons (H\u003csub\u003ec\u003c/sub\u003e, H\u003csub\u003ed\u003c/sub\u003e, and H\u003csub\u003ee\u003c/sub\u003e) exerted an up-field shift upon increase CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] concentration from 0 to 0.5 equivalent, indicating the tight encapsulation of ethylene pyridine moiety by CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] that moved from the phenylpyridine unit (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and Supplementary Figs.\u0026nbsp;25\u0026ndash;26). Moreover, in the 2D NOESY spectrum of the TPE-PY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] assembly (Supplementary Fig.\u0026nbsp;27), we could easily locate the cross-peaks between the protons of vinyl functional groups and CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The 2:1 stoichiometry ratio obtained from Job\u0026rsquo;s plot (Supplementary Fig.\u0026nbsp;28) and the strong binding constant of 2.90\u0026times;10\u003csup\u003e12\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e for TPE-PY/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) provided further support for the aforementioned findings. These findings suggested that the vinylpyridine and phenyl pyridines moieties of TPE-PY were included in the cavities of CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], respectively, ultimately generating a ternary supramolecular assembly with a stoichiometric ratio of TPE- PY:CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]:CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u0026thinsp;=\u0026thinsp;2:2:1. Thus, we deduced that the co-assembly of TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] went through a similar assembly process to form a linear supramolecular aggregate with a stoichiometric ratio of 1:2:1. From the above experimental results, it can be seen that CB[n] (n\u0026thinsp;=\u0026thinsp;7, 8) possesses a different binding affinity to TPE-DPY, leading to a diverse topological morphology for the supramolecular assembly. Profited by the host-guest complexation, hydrophobic interaction, and π \u0026ndash; π stacking interactions, the binary assembly of TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] presents spherical nanoparticles with adjustable dimensions, and CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] with a larger hydrophobic cavity binds with TPE-DPY to form a n:n rod-shaped pseudorotaxane. The ternary co-assembly TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] has a more robust rigid structure than TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and TPE-DPY/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], resulting in a linear self-assembly stacked multi-layered three-dimensional nanoplates.\u003c/p\u003e\u003cp\u003eSubsequently, the configuration-confined photophysical properties of the assembly of TPE-DPY and CB[n] (n\u0026thinsp;=\u0026thinsp;7/8) were explored. With the binary assembly of CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] or CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], the absorption peak of TPE-DPY redshifted from 315 nm to 320 nm and 330 nm, respectively, and a comparable red shift by 13 nm occurred in the co-assembly of CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). For the photoluminescence spectra shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, the guest molecule TPE-DPY exhibited a fluorescence emission at 390 nm with an excitation of 315 nm, and no phosphorescent signal was captured in the delayed spectrum. The assembly of TPE-DPY/2CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], TPE-DPY/4CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], TPE-DPY/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] displayed a remarkable new emission peak around 530 nm as compared to the free TPE-DPY (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Differing from the steady-state PL spectrum spectra, the delay spectra of these assemblies showed a major emission peak near 530 nm, illustrating its long-lived feature, which was further proved by their microsecond lifetime obtained by the time decay curve measurement (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, c). Notably, in contrast to the binary assembly TPE-DPY/2CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], TPE-DPY/4CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and TPE-DPY/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], the ternary assembly TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] had a stronger luminous intensity with extending the lifetime from 8.29 \u0026micro;s to 59.36 \u0026micro;s (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), because of the synergistic confinement effect of CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] on guest molecules and the supramolecular nanostructure formed by the linear rigid assembly layer by layer enabling valid shielding effect on the quencher. Additionally, after the injection of Ar, the lifetime of TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] aqueous solution at 540 nm was significantly increased from 59.36 \u0026micro;s to 129.97 \u0026micro;s (Supplementary Fig.\u0026nbsp;30) due to the avoidance of the triplet electron quenching caused by oxygen, further confirming the phosphorescence properties of emission peak at 540 nm. The above experiment results demonstrated that the macrocyclic confinement can effectively induce a phosphorescence emission, and the topological morphology of supramolecule assembly can be regulated by adjusting the ratios of CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], presenting different photophysical properties.\u003c/p\u003e\u003cp\u003eOn the basis of the binary supramolecular assembly, the multivalent cascade assembly has evolved into an effective method to improve phosphorescence performance.\u003csup\u003e42\u003c/sup\u003e Herein, HACD as a polysaccharide targeting agent has been introduced into TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] to construct a secondary assembly, which resulted in an exchanged topology from hierarchical self-assembled nanoplates to spherical nanoparticles (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Dynamic light scattering (DLS), TEM, and zeta potential experiments were carried out to explore the assembly behavior for TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]@HACD. DLS measurements suggested that TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]@HACD assembly had an average hydrodynamic diameter of 236 nm, which matched the size of nanospheres in the TEM image (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). Moreover, on the contrary of TPE-DPY and TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] that possessed a positive zeta potential at +\u0026thinsp;1.74 and +\u0026thinsp;1.80 mV, respectively, a negative potential value of TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]@HACD was obtained as -0.335 mV (Supplementary Fig.\u0026nbsp;31), revealing the successful construction of the multicomponent assembly. Remarkably, the cascade assembly of TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]@HACD not only changed the topological morphology but also achieved a HACD-activated single-molecular PRET based on macrocyclic confinement. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef and Supplementary Fig.\u0026nbsp;32, upon the addition of HACD, the assembly of TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] excited by 333 nm showed a weak phosphorescence at 535 nm with a lifetime of 69.83 \u0026micro;s, and a dominant emission band centered at 700 nm with a lifetime of 21.60 \u0026micro;s, ascribing to the delayed fluorescence of the methoxytetraphenyl-vinylpyridine part stimulated by PRET.\u003c/p\u003e \u003cp\u003eTo further confirm the multivalent cascade confined single-molecular PRET luminescence behavior, a series of control experiments have been performed. The reference molecule PY-1 displayed CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]-induced strong RTP emission around 520 nm with a lifetime of 388.20 \u0026micro;s (Supplementary Fig.\u0026nbsp;33), suggesting that the phosphorescence emission of the TPE-DPY assembly emanated from the phenylpyridine units. The steady-state PL spectrum of TPE-1/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] excited by 450 nm presented an emission peak at 720 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed) with a nanosecond lifetime measured as 2.74 ns, revealing the pure fluorescence properties of methoxy tetraphenylvinylpyridine unit (Supplementary Fig.\u0026nbsp;34). Similarly, under the excitation of 450 nm, TPE-DPY, TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]@HACD showed a fluorescence emission of 720 nm, where the lifetime was measured as 0.71 ns, 0.74 ns and 0.85 ns, respectively. No emission signal was obtained in the delay spectrum, verifying the property of HACD-mediated NIR delayed fluorescence at 700 nm that was excited by 333 nm (Supplementary Fig.\u0026nbsp;35). Furthermore, a large overlap was captured between the absorption spectra of TPE-1/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and the phosphorescence spectra of PY-1/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), which provided a prerequisite for the PRET derived from phenyl pyridines unit to methoxy-tetraphenylethylene portion within a single-molecule. It is worth noting that no effective PRET phenomenon was obtained in the HACD-assembly doping system with PY-1/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] as the donor and TPE-1/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] as the acceptor, which may be caused by the uncontrollable large distance between the donor and the acceptor without covalent connection, hindering the generation of the energy transfer process (Supplementary Fig.\u0026nbsp;36). Moreover, the addition of bare HA into the TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] solution cannot activate an efficient PRET like HACD, highlighting the important role of β-CD in this cascade assembly process (Supplementary Fig.\u0026nbsp;37). Accordingly, the binding behavior between the reference TPE-1 and β-CD was investigated by \u003csup\u003e1\u003c/sup\u003eH NMR spectra. It was shown that upon the addition of β-CD, the protons on methoxyphenyl (H\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e, H\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e) in TPE-1 shifted slightly to low-field, while the protons in styryl pyridiniums remained unchanged (Supplementary Fig. S38), indicating the complexation of β-CD and methoxyphenyl unit. and the association constant of TPE-1/β-CD was determined to be 344.09 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;39). These experimental results consistently indicated that the anion effect of HA and the encapsulation of β-CD to methoxy-tetraphenylethylene moiety contributed to the reconstruction of topological morphology for TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], which facilitated the single intramolecular PRET process leading to 700 nm NIR delayed fluorescence emission with large Stokes shift of 367 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Commonly, the phosphorescence spectra of the assemblies TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]@HACD and TPE-DPY/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]@HACD also exhibited NIR emission peaks at 700 nm, implying the universality of HACD activation for single-molecular PRET process (Supplementary Fig.\u0026nbsp;40).\u003c/p\u003e\u003cp\u003eIn order to explore the application of macrocyclic confinement and HACD-activated single-molecule PRET system, cell imaging experiments were constructed. First, Human cervical carcinoma cells (Hela cells) and human embryonic kidney cells (293T cells) were treated with TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]@HACD for 12h, respectively, and then incubated with Hoechst and Mito-Tracker Green for localization experiment. The confocal laser scanning microscopy (CLSM) experiments were performed to investigate the intracellular NIR emission signals. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, Hela cells exhibited a bright NIR luminescence in a red channel (650\u0026ndash;750 nm), whereas almost no red emission signal was found for normal 293T cells. These imaging results implied that TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]@HACD was preferentially internalized by cancer cells rather than normal cells, which may be caused by the overexpressed HA receptors for cancer cells. Furthermore, colocalization analysis demonstrated that the NIR luminescence signal overlapped well with the green signal of Mito Tracker, which corresponded to the yellow region in the merged image (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). It revealed the ability of TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]@HACD for targeted mitochondria imaging in cancer cells, and the high Pearson correlation provided strong evidence for this result (Supplementary Fig.\u0026nbsp;41). Finally, CCK-8 assays were conducted to evaluate cytotoxicity experiments on the above two cells, and the high survival rate indicated low cytotoxicity of the assembly TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]@HACD (Supplementary Fig.\u0026nbsp;42).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn summary, a single-molecule PRET is activated by the macrocyclic confinement of CB[n] (n\u0026thinsp;=\u0026thinsp;7,8) and the secondary assembly of HACD to achieve an NIR-delayed fluorescence emission. Based on the different cavity sizes, the primary assembly of CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] to TPE-DPY presented a macrocyclic confinement-induced phosphorescence behavior accompanied by controllable topological morphologies, which realized a transformation from nanosphere, rod-shaped pseudorotaxane to hierarchical self-assembled nanoplate. Especially, the secondary assembly of HACD activated the single intramolecular PRET from the phenyl pyridines unit to the methoxy-tetraphenylethylene portion, generating an NIR delayed fluorescence emission at 700 nm. Different from the dye-doped PRET system, this macrocyclic-confinement polysaccharide activated single molecular PRET system displayed a large Stokes shift of 367 nm and was successfully applied in mitochondrial-targeted imaging for cancer cells, which provides a new approach for the construction and application of single-molecule PRET.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eMaterials.\u003c/b\u003e Except for additional stated, all reagents and solvents were available from commercial sources and used directly without any purification. HA-CD that we used was synthesized on the basis of the literature, indicating the degree of substitution (DS) was 16.43%. \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR spectrums were recorded through Bruker AV400. Two-dimensional NMR (COSY, DOSY, NOESY, and ROESY) spectra were measured on Bruker AVANCE III HD 400 spectrometer. High-resolution mass spectrometry (HR-MS) was recorded on a Q-TOF LC-MS in an Electrospray ionization (ESI) source. UV-vis absorption was kept details on Shimadzu UV-3600 spectrophotometer with a PTC-348WI temperature controller at 298 K. Photoluminescence (PL) spectrum and time-correlated decay profiles were documented on Edinburgh Instruments FS5 (Livingstone, UK). The TEM experiment was carried out on FEI Tecnai G2 F20 under 200 KV. SEM was accomplished on FEI Apreo S LoVac scanning electronic microscope working at an accelerating voltage of 30 keV. The Zeta potentials were examined on Brookhaven ZETAPALS/BI-200SM at 298 K. Dynamic Light Scattering (DLS) was determined by using a laser lights-scattering spectrometer (BI-200SM) equipped with a digital correlator (Turbo Corr) at 635 nm at a scattering angle of 90\u0026deg;. Cell images were captured on Olympus FV1000 Laser scanning confocal microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCytotoxicity experiments.\u003c/b\u003e The cells, including Hela cells and 293T cells, were all gained from the Cell Resource Center of China Academy of Medical Science in Beijing. These cells were cultured in particular conditions with the addition of 10% FBS and 1% penicillin/streptomycin in the DMEM nutrient medium and humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere at 37 ℃. The Hela cells and 293T cells were incubated with TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]@HACD at distinct concentrations in 96-well plates for 24 hours. The relative cellular viability was determined by the CCK8 assay.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell imaging experiments.\u003c/b\u003e The Hela cells and 293T cells were precultured in confocal petri dishes, respectively, for 24 h. Then, the well-cultured cells were incubated with TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]@HACD (20 \u0026micro;M) for a further 2 h. The cells were stained with Hoechst (1 nM) and Mito-Tracker Green (1 nM) for half an hour, washed three times with PBS, and then added PBS (1 ml) to observe by microscope.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthesis of compound TPE-DPY.\u003c/b\u003e Under N\u003csub\u003e2\u003c/sub\u003e protection, 4,4\u0026prime;-[[2,2-bis(4-methoxyphenyl)ethenylidene]bis(4,1-phenylene-2,1-ethenediyl)]pyridine (54 mg, 0.09 mmol) and PY-1 (94.5 mg, 0.22 mmol) were dissolved in CH\u003csub\u003e3\u003c/sub\u003eCN (5 ml). The reaction mixture was heated to 85 ℃ for 36 hours. After the reaction, CH\u003csub\u003e3\u003c/sub\u003eCN was evaporated, and acetone was used for ultrasound cleaning. Then, the mixture was filtered and washed with acetone twice. Additionally, the crude powder was purified by heat filtration and recrystallization to give an orange solid (18 mg, yield: 13.6%). \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, Methanol-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e) δ 9.07 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.7 Hz, 4H), 8.86 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.5 Hz, 4H), 8.49 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.5 Hz, 4H), 8.20 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.6 Hz, 4H), 7.97 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5 Hz, 4H), 7.91 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.2 Hz, 2H), 7.84 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5 Hz, 4H), 7.56 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 4H), 7.39 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.2 Hz, 2H), 7.15 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2 Hz, 4H), 6.98 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.6 Hz, 4H), 6.72 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.7 Hz, 4H), 4.84 (s, 4H), 4.76 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5 Hz, 4H), 3.75 (s, 6H), 2.84\u0026ndash;2.78 (m, 4H).; \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, Methanol-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e4\u003c/sub\u003e) δ 158.97, 155.76, 154.58, 146.95, 144.83, 143.98, 141.73, 135.52, 133.19, 132.80, 132.42, 131.95, 129.61, 127.80, 127.00, 124.92, 124.01, 122.16, 112.94, 57.27, 56.79, 54.26, 31.99.; HRMS (ESI) m/z for C\u003csub\u003e70\u003c/sub\u003eH\u003csub\u003e62\u003c/sub\u003eBr\u003csub\u003e6\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e calcd. [M-4Br]\u003csup\u003e4+\u003c/sup\u003e 287.5799, found: 287.0806.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eData supporting the findings of this study are included within the article and Supplementary Information files. Data are available from the authors upon request.\u003c/p\u003e \u003c/div\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eX.Z. and X.B. contributed equally to this work and were primarily responsible for the experiments, then measured and analyzed the experiment data. X.Z. wrote the manuscript. H-Y Z. and L-H W. gave valuable suggestions. Y.L. supervised the work and edited the manuscript. All authors analyzed and discussed the results and reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe thank National Nature Science Foundation of China (NNSFC, Grant Nos. 22131008 and 22271165, China Fundamental Research Funds for the Central Universities and the Haihe Laboratory of Sustainable Chemical Transformations for financial support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePrins LJ, Reinhoudt DN, Timmerman P (2001) Noncovalent synthesis using hydrogen bonding. Angew Chem Int Ed 40:2382\u0026ndash;2426\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMetrangolo P, Meyer F, Pilati T, Resnati G (2008) Terraneo. Halogen bonding in supramolecular chemistry. 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Angew Chem Int Ed 60:27171\u0026ndash;27177\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Room-temperature phosphorescence, phosphorescence resonance energy transfer, large Stokes shift, near-infrared emission, cell imaging","lastPublishedDoi":"10.21203/rs.3.rs-3784778/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3784778/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA single-molecule phosphorescence resonance energy transfer (PRET) system with a large Stokes shift of 367 nm and near-infrared (NIR) emission is constructed by alkyl-bridged methoxy-tetraphenylethylene-phenylpyridines derivative (TPE-DPY), cucurbit[n]uril (CB[n], n\u0026thinsp;=\u0026thinsp;7,8), and β-cyclodextrin modified hyaluronic acid (HACD). The experiment results demonstrate that the high binding affinity and various stoichiometric ratios of CB[n] (n\u0026thinsp;=\u0026thinsp;7, 8) to TPE-DPY not only regulate the topological morphology of supramolecular assembly but also induce different phosphorescence emissions. The assembly of TPE-DPY and CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] presents spherical nanoparticles, exhibiting an emerging phosphorescence emission at 525 nm via the macrocyclic confinement effect to phenyl-pyridine units. CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] with a larger hydrophobic cavity binds with TPE-DPY to form an n:n pseudorotaxane nanorod, which induces an efficient phosphorescence at 545 nm. Varying from the binary assembly of CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] or CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], an entirely distinct topological organic three-dimensional nanoplate is obtained by the co-assembly TPE-DPY with CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], accompanying enhanced phosphorescence at 540 nm. Uncommonly, the secondary assembly of HACD and TPE-DPY/CB[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]/CB[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] activates a single intramolecular PRET process derived from phenyl pyridines unit to methoxy-tetraphenylethylene function group, enabling an NIR delayed fluorescence at 700 nm excited by 333 nm, which ultimately applied to mitochondrial targeted imaging for cancer cells.\u003c/p\u003e","manuscriptTitle":"Single-Molecule Phosphorescence Resonance Energy Transfer for NIR Targeted Cell Imaging","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-08 17:34:17","doi":"10.21203/rs.3.rs-3784778/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a74ed0cd-b54a-4084-8af5-333734bb6c77","owner":[],"postedDate":"January 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":27666897,"name":"Physical sciences/Chemistry/Supramolecular chemistry/Self-assembly"},{"id":27666898,"name":"Physical sciences/Chemistry/Supramolecular chemistry"}],"tags":[],"updatedAt":"2024-06-06T07:07:46+00:00","versionOfRecord":{"articleIdentity":"rs-3784778","link":"https://doi.org/10.1038/s41467-024-49238-5","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2024-06-05 04:00:00","publishedOnDateReadable":"June 5th, 2024"},"versionCreatedAt":"2024-01-08 17:34:17","video":"","vorDoi":"10.1038/s41467-024-49238-5","vorDoiUrl":"https://doi.org/10.1038/s41467-024-49238-5","workflowStages":[]},"version":"v1","identity":"rs-3784778","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3784778","identity":"rs-3784778","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}