Highly two‐photon and X‐ray excited long‐persistent luminescence in a crystalline host‐guest aggregate

As a unique type of supramolecular self‐assemblies, crystalline host‐guest aggregates have attracted extensive interests in multiple application fields. Herein, a crystalline host‐guest aggregate LIFM‐HG1 was obtained with curcubit[8]uril as the host and carboxypyridinium salt as the guest. Single‐crystal structural analysis indicates that the presence of abundant weak interactions in LIFM‐HG1 provides a rigid environment for the guest molecule and effectively blocks the external quenchers. Spectral analysis and theoretical calculations confirm the presence of robust triplet energy levels in LIFM‐HG1. Even more impressively, the intersystem crossing channels of the guest molecules are greatly opened up after the formation of the crystalline host‐guest aggregate, resulting in a large kisc of 6.70 × 107 s−1 at room temperature for LIFM‐HG1 (which is ∼0 for pure guest), leading to fascinating multichannel (including one‐photon, two‐photon, and X‐ray) excited LPL properties. In addition, the crystalline LIFM‐HG1 has a much higher triplet state luminescence efficiency under X‐ray and two‐photon excitation than that under single‐photon excitation (AP/AF = 86.8, 44.8, 10.7 under the three circumstances, respectively). And the phosphorescent emission intensity of LIFM‐HG1 is 27.6 times higher than that of the crystalline guest under X‐ray excitation. As a result, LIFM‐HG1 shows a long afterglow retention time under both single‐ and two‐photon excitation, and an impressive afterglow retention time of 1 s under X‐ray excitation. Furthermore, the excellent lysosomal targeting and low cytotoxicity by the formation of host‐guest aggregate makes LIFM‐HG1 promising to be used as a novel lysosomal‐targeted two‐photon excited phosphorescent tracer.


INTRODUCTION
[20] Practically, most organic molecules fail to exhibit significant room temperature phosphorescence or afterglow luminescence.This is mainly caused by the following factors: (1) slow intersystem crossing (ISC) of excited electrons between the singlet and triplet energy levels in organic molecules, resulting in a low probability of triplet state electron population, (2) complex and frequent molecular motions and bond vibrations as well as perturbations in the environment cause rapid dissipation of the triplet state energy, (3) the isolated states and loose stacking allow external quenchers to destroy triplet excitons easily.The presence of these obstacles makes the design and application of efficient organic LPL materials very difficult.
As a supramolecular container with unique nano-cavities, cucurbit [n]uril is widely used to encapsulate a variety of organic or inorganic components to obtain materials with specific functions. [21,22]25][26] On the one hand, the confinement effect of the host cavity sufficiently inhibits the motion of the guest molecules and/or the vibration of the chemical bonds, which makes the nonradiation deactivation pathway inefficient.On the other hand, the explicit aggregation mode and the dense stacking model make the exploration of the structure-property relationships of the material easier, and the small degree of pore freedom can also afford further resistance to external quenchers.[29] This is due to special requirements for the guest molecules, such as their size and charge must be matched, the luminescence needs to be efficient, and their energy level distribution needs to be appropriate, and so on.These barriers have severely limited the development of crystalline host-guest aggregates with LPL activity.Therefore, more efforts are needed in the future to explore crystalline host-guest aggregates with long-persistent luminescence.
At present, most organic systems with LPL properties exhibit a single excitation channel.The reports of organic LPL materials with multiple excitation channels are still relatively scarce.Single excitation mode (such as single-photon excitation) and inflexible regulation characteristics make single-channel excited LPL materials unable to meet the growing demand for practical applications.[32] The main excitation modes that have been reported are singlephoton excitation, [33] two-photon excitation, [34,35] X-ray excitation, [36,37] ultrasonic excitation [38,39] and mechanical force excitation, etc. [40] Among them, single-photon excitation is the most familiar excitation mode with positive Stocks shifts and a well-defined luminescence mechanism (Figure 1A) as its typical features.In contrast, two-photon excitation as a unique nonlinear photon absorption behavior has aroused a strong desire to explore. [41,42]Due to the small excitation energy, deep tissue penetration, high energy conversion efficiency and amazing luminescence mechanism (Figure 1B), the two-photon excited luminescence materials are widely used in bio-imaging, [43] photodynamic therapy, [44,45] pressure sensors, [46] white light emitting devices, and many other fields. [47]In addition, high-energy X-ray was also used as a special excitation source. [48]As shown in Figure 1C, when a sample is bombarded by highenergy photons (X-rays), a part of the photon energy is radiated as X-rays by means of Compton scattering, [49] and the other part is absorbed by the inner electrons outside the nucleus, resulting in a photoelectric effect and the production of high-energy escaping electrons and deep holes. [50]nd then, the electron-hole pairs are gradually generated under thermalization and further recombined to produce 25% singlet excitons and 75% triplet excitons (spin statistical rule). [49,50]Therefore, theoretically, X-ray excitation has higher triplet luminescence efficiency.Herein, we prepared a crystalline host-guest aggregate LIFM-HG1 (LIFM stands for Lehn Institute of Functional Materials, H stands for host, and G stands for guest) with long-persistent luminescence from multichannel excitation by a one-pot solvothermal reaction using curcubit [8]uril as the host and carboxypyridinium salt as the guest (Figure 2A).And the photon absorption and emission behaviors in LIFM-HG1 were systematically explored.The abundant weak interactions and dense stacking structure provide a favorable rigid environment for the guest molecule and also block the intrusion of external quenchers.Spectral analysis and theoretical calculations confirm the existence of the robust triplet energy levels in LIFM-HG1.Interestingly, LIFM-HG1 can be excited by one-and two-photon as well as X-ray and emits green long-persistent luminescence.To the best of our knowledge, this is the first case of a crystalline host-guest aggregate with long-persistent luminescence by multi-channel excitation.And furthermore, the excellent lysosomal targeting and low cytotoxicity make LIFM-HG1 promising to be used as a novel lysosomal-targeted phosphorescent tracer.

RESULTS AND DISCUSSION
The crystalline host-guest aggregate LIFM-HG1 was prepared by a one-pot solvothermal synthesis method (see Supporting Information for details).A series of characterizations such as NMR, HPLC, FT-IR, UV-Vis absorption spectrum, and the powder X-ray diffraction patterns (PXRD) indicated that LIFM-HG1 has high purity and crystallinity (Figures S1-S8).Thermogravimetric analysis and in situ PXRD analysis showed that LIFM-HG1 has high thermal stability (578 K, Figures S9 and S10).Single-crystal X-ray diffraction analysis (Table S1) showed that guest (G), curcubit [8]uril (H) and LIFM-HG1 were crystallized in the P2 1 /n, I4 1 /a, and Pbca space groups, respectively.As shown in Figure S10a, a 2:1 (G:H) type of ternary binding mode is identified in the LIFM-HG1 crystal.A curcubit [8]uril cavity contains two G molecules, which are inversely parallel to each other.The abundant hydrogen bonding interactions (2.31-3.80Å) and π⋅⋅⋅π interactions (∼3.77Å) in LIFM-HG1 allow the G molecules to be firmly anchored in the nanocavities of curcubit [8]uril and form a dense stacking model.This result was further demonstrated by the IGMH (independent gradient model based on Hirshfeld partition) calculations (Figure 2B and Figure S11) and Hirshfeld surfaces analysis (Figure 2D and Figure S12).The electrostatic potential (ESP) surface reveals the charge distribution on its molecular surface (Figure S13).The small free volume (Figure S14) facilitates resistance to external quenchers, such as O 2 , etc. [51] To explore the assembly behavior of LIFM-HG1 in aqueous solution, NMR, UV-Vis, fluorescence titration spectroscopy, ITC and 2D NMR spectroscopy were tested (Figures S15-S19, Table S2).The G molecule was determined to be encapsulated into the nano-cavity of curcubit [8]uril and formed a ternary complex in a 2:1 (G:H) binding mode.And the results were consistent with the single crystal structure of LIFM-HG1.
The photoluminescence spectrum of the crystalline hostguest aggregate LIFM-HG1 is shown in Figure 3A.A significant excitation band located at 337 nm can be observed.And the photoluminescence spectrum of LIFM-HG1 shows multiple emission bands.The emission band with a shorter lifetime (τ 398nm = 1.91 ns) is referred to its fluorescence emission (Figure S20a and Table S3).And the emission band with a longer lifetime (τ 504nm = 327.72 ms) is designated as its phosphorescence emission band (Figure S20b and Table S4).The appearance of multiple peaks in the fluorescence emission band in the prompt emission spectrum may be due to the scattering of light by LIFM-HG1 crystal particles.After grinding the crystalline LIFM-HG1 (Figure S21), its fluorescence emission band becomes a wide peak (Figure S22).It indicates that the multiple fluorescence peaks of crystalline LIFM-HG1 are mainly caused by the scattering effect of crystal particles.In addition, the luminescence of LIFM-HG1 remains the same as under the bulk crystal (Figures S21 and S22).It is well known that low temperature favors the suppression of non-radiative decay channels, resulting in enhanced luminescence lifetimes and quantum yields.As a result, the fluorescence lifetime of LIFM-HG1 was enhanced 10-fold at 77 K (Figure S20c and Table S5), and its phosphorescence lifetime was enhanced four-fold (Figure S20d and Table S6).In addition, the quantum yield of LIFM-HG1 at 77 K was also significantly enhanced (from 14.0% to 62.4%) (Table S7).A problem that cannot be ignored is the source of luminescence of LIFM-HG1.As shown in Figures S23 and  S24, dilute solutions of the guest (G) have similar fluorescence emission bands as the crystalline LIFM-HG1 (Table S8-S9).And, the phosphorescence emission band similar to that of LIFM-HG1 can also be captured in the crystalline G (Table S10-S14).Therefore, the guest molecule in LIFM-HG1 is designated as its main luminescence center.
Interestingly, the radiative decay channels of G are significantly enhanced and the nonradiative decay channels are strongly suppressed after the formation of the host-guest aggregate (Figure 4A, Figure S25 and Tables S15 and S16).Moreover, the intersystem crossing rates (k isc ) of crystalline LIFM-HG1 reached 6.70 × 10 7 s −1 (RT) and 1.77 × 10 7 s −1 (77 K), respectively.This result is much larger than that of crystalline G (Figure 4B).Encouragingly, the dramatically increased luminescence lifetimes and quantum yields have been observed (Figure 4C,D and Figure S26).Compared with crystalline G, the phosphorescence lifetime of LIFM-HG1 is 747.8 (RT) and 236.2 (77 K) times longer (Figure 4C), and its total luminescence quantum yield is more than 23 times (Figure 4D).As shown in Figures 3B,C and 5A, the crystalline host-guest aggregate LIFM-HG1 exhibits a unique single-photon excited long-persistent luminescence phenomenon.Bright green afterglow emission can be observed when LIFM-HG1 is irradiated with a suitable excitation source (Video S1).At low temperature, the afterglow retention time of LIFM-HG1 can reach an amazing 15 s.Even at room temperature, the afterglow retention time of LIFM-HG1 can reach about 5 s.In addition, the green afterglow of LIFM-HG1 can still be observed at a high temperature of 337 K.And then, different excitation wavelengths were used to investigate the behavior of the excited states in LIFM-HG1.Similarly, significant green afterglow could be captured (Figure S27-S36).
Excitingly, the crystalline host-guest aggregate LIFM-HG1 also responds well to the two-photon excitation source.As shown in Figure 3D, bright green phosphorescence emission is observed when LIFM-HG1 is irradiated with a two-photon excitation source.And the emission spectrum is consistent with that when single-photon excitation is applied (Figure S37).Crystalline LIFM-HG1 has the maximum emission intensity and two-photon excited phosphorescence (TPEP) cross section (σ = 94.10GM) when using a 670-nm excitation source (Figures 3E and 4E).The small threshold (47.30μJ) and large two-photon absorption (TPA) cross section (735.13GM) indicate that the crystalline host-guest aggregate LIFM-HG1 has an agile response to the two-photon excitation source (Figure S38).In comparison, the crystalline G has only a small TPEP cross section (σ = 5.04 GM) due to its weaker phosphorescence (Figure S39).Encouragingly, the long-persistent luminescence of the two-photon excitation was captured flexibly in LIFM-HG1 (Figures 3F and 5B, and Video S2).The afterglow spectrum demonstrated the significant afterglow emission of LIFM-HG1 in the two-photon excitation range of 560-750 nm (Figures S40 and S41).At low temperature, the afterglow retention time (∼15 s, Figure 5B) for two-photon excitation can reach the same level as that for single-photon excitation.In addition, the tolerance limit of long-persistent luminescence for two-photon excitation of LIFM-HG1 reaches 337 K, which is the same as the result under single-photon excitation (Figures S42 and S43).
Importantly, X-ray was used as an excitation source to investigate the triplet excited state behavior of LIFM-HG1.As shown in Figure 3G, the crystalline guest (G), curcu-bit [8]uril (H) and LIFM-HG1 were placed in the X-ray atmosphere.And the steady-state emission spectrum of all three were also collected.Interestingly, LIFM-HG1 exhibits strong X-ray excited phosphorescence (XEP) emission, while the luminescence of the other materials is very weak.The luminescence of the guest is enhanced by a factor of 27.6 after the formation of the crystalline host-guest aggregate (Figure 4F).It suggests that the confined cavity of the host can strongly suppress the nonradiative deactivation pathway of the guest molecule, thus stabilizing the triplet exciton for efficient long-persistent luminescence.In addition, the phosphorescence intensity of LIFM-HG1 is enhanced with increasing X-ray dose (Figure 3H,I).An excellent linear relationship between phosphorescence intensity and X-ray dose was obtained by fitting (Figure S44), which makes LIFM-HG1 promising for the detection of harmful high-energy rays.And then, the stability of crystalline LIFM-HG1 for X-ray was investigated.As shown in Figure S45, the luminescence intensity of LIFM-HG1 does not change significantly with time at the same X-ray output.And after 100 on-off cycles, the luminescence intensity of LIFM-HG1 changes very little (Figure S46).It indicates that the crystalline LIFM-HG1 has good stability to X-ray.And the crystalline LIFM-HG1 also has a low absorption and detection limit (12.81 mGy/s) for X-ray (Figures S47 and S48).Excitingly, LIFM-HG1 has an extremely long luminescence lifetime of 428.81 ms under X-ray excitation (Figure S49), and this value is 15.3 times that of crystalline G (Figure 4F).Moreover, the crystalline LIFM-HG1 has a much higher triplet state luminescence efficiency under X-ray and two-photon excitation than that under single-photon excitation (A P /A F = 86.8,44.8, 10.7 under the three circumstances, respectively, Figure 4 G).As a consequence, it reveals that the crystalline LIFM-HG1 has an afterglow retention time of more than 1 s under X-ray excitation (Video S3).This is the maximum of the crystalline host-guest materials reported so far.
Time-dependent density functional theory calculations were used to investigate the electronic structure and excited state behavior of the aggregates (see Supporting Information for details).The geometry optimization and electronic structure natures of guest (G) dimer, curcubit [8]uril (H) and LIFM-HG1 at the level of B3LYP-GD3(BJ)/6-311G** have been evaluated.Considering the abundant weak interactions in the LIFM-HG1 crystal, a QM/MM model based on the Xray single-crystal structure was established (Figure S50).The electronic transition characteristics of the excited states (S 1 and T 1 ) of the aggregates show a significant dimeric behavior (Figures S51-S53).In addition, the calculated fluorescence emission spectrums were obtained in good agreement with the experimental ones (Figure S54).The energy level distribution obtained from theoretical calculations indicates that the guest has an excited state energy level structure similar to that of LIFM-HG1 (Figure 2E, Figure S55, Tables S17-S24).Therefore, this further proves that the G dimer in LIFM-HG1 is the main source of luminescence.And the large spin-orbital coupling matrix elements (SOCME, ξ) between S 1 and T 1 also indicates the presence of an effi-cient intersystem crossing (ISC) process (Tables S25-S27).Moreover, the triplet spin density distributions (TSDD) of G dimer, curcubit [8]uril (H) and LIFM-HG1 were calculated and are shown in Figure 2C and Figure S56.The results indicate that the C and N elements on the benzene and pyridine rings are the main carriers of their spin single electrons.
Furthermore, the crystalline LIFM-HG1 was used in cell imaging based on its excellent single-and two-photon excited phosphorescence properties.The luminescent properties of LIFM-HG1 in aqueous solution are also shown in Figure S57.As a result, LIFM-HG1 was still able to emit the same green phosphorescence as in the crystalline state after being sonicated in water.As shown in Figure 6A, LIFM-HG1 can be well taken up by HeLa cells and emits green phosphorescence.Compared to the G and G+H solution assembly systems, crystalline LIFM-HG1 has more significant green phosphorescence emission (Figure S58).This result suggests that crystalline LIFM-HG1 has greater cell imaging potential than the solution-assembled systems (G+H) and G.And then, the one-and two-photon microscopic imaging results further proved the excellent cellular uptake of crystalline LIFM-HG1 (Figure 6B).In order to explore the action sites of the aggregate, the co-localization experiments were developed (Figure 6C and 6D).The results show that crystalline LIFM-HG1 is well enriched in the lysosomes of cells.In addition, the low cytotoxicity allowed for increased possibilities of practical applications of crystalline LIFM-HG1 (Figure S59).Similar results have been observed in other cells as well (Figure S60 and S61).Therefore, the crystalline LIFM-HG1 is expected to be used as a novel lysosomal-targeted phosphorescent chromophore.

CONCLUSION
In summary, we prepared a crystalline host-guest aggregate LIFM-HG1 with long-persistent luminescence with multi-channel excitation by a one-pot solvothermal reaction using curcubit [8]uril as the host and carboxypyridinium salt as the guest.Crystal structure analysis and IGMH calculations indicate the presence of abundant weak interactions in LIFM-HG1.These weak van der Waals interactions not only restrict the guest molecule to a rigid environment, but also effectively stabilize the triplet exciton.Spectral analysis and theoretical calculations suggest that the long-lived phosphorescence emission in LIFM-HG1 is derived from its well-defined triplet state energy level.Deeper study reveals that the formation of the crystalline host-guest ensemble greatly opens up the intersystem crossing channels of the guest molecule, allowing the kisc of LIFM-HG1 to reach an impressive 6.70 × 10 7 s −1 (RT) and 1.77 × 10 7 s −1 (77 K).As a result, LIFM-HG1 can be excited by single-photon, two-photon and X-ray to emit bright green long-persistent luminescence.And the longer afterglow retention times were collected under single-photon, two-photon and X-ray excita-tion.Moreover, there is a 747.8-fold (RT) and 236.2-fold (77 K) increase in the phosphorescence lifetime of LIFM-HG1 compared to the crystalline guest, and its total luminescence quantum yield is increased by more than 23-fold.The phosphorescent emission intensity of LIFM-HG1 is 27.6 times higher than that of the crystalline guest under X-ray excitation.And then, LIFM-HG1 was used in cellular imaging, which has excellent lysosomal targeting and low cytotoxicity.These results provide favorable guidance for the design and synthesis of crystalline host-guest aggregates with multichannel excitation for long-persistent luminescence, and are also expected to serve as a novel phosphorescent tracer for harmful high-energy rays and lysosomal targeting.

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I G U R E 2 (A) The molecular structure of guest (G) and curcubit[8]uril (Host).(B) The isosurfaces of independent gradient model based on Hirshfeld partition (IGMH) of LIFM-HG1.(C) The triplet spin density distribution of LIFM-HG1 calculated at the optimized T 1 geometry (The curcubit[8]uril molecular was omitted for clarity, isovalue = 0.005).(D) Hirshfeld surface analysis of G dimer in LIFM-HG1 mapped with the parameter d norm along with 2D fingerprint plots with a specific interaction.(E) The calculated energy level diagrams and spin-orbital coupling matrix elements (SOCME, ξ) of G dimer and LIFM-HG1 at the B3LYP-GD3(BJ)/6-311G** level.

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I G U R E 3 (A-C) One-photon luminescence: (A) The excitation, prompt, and delayed emission spectrum of crystalline LIFM-HG1 at room temperature (delayed time = 1 ms); (B) The excitation-delayed emission mapping of crystalline LIFM-HG1 at room temperature; (C) Afterglow spectrum of crystalline LIFM-HG1 at different time intervals after excitation by 310 nm at room temperature.(D-F) Two-photon luminescence: (D) The emission spectrum of crystalline LIFM-HG1 excited by a two-photon source at room temperature; (E) The two-photon excited phosphorescence (TPEP) cross sections (σ) and phosphorescence intensity at 504 nm of crystalline LIFM-HG1 at room temperature; (F) Afterglow spectrum of crystalline LIFM-HG1 at different time intervals after excitation by 670 nm at room temperature.(G-I) X-ray luminescence: (G) The emission spectrum of guest (G), curcubit[8]uril (H) and LIFM-HG1 under the irradiation of X-ray (power: 9.6 W; dose rate: 982.47 mGy/s); (H) the prompt emission spectrum of crystalline LIFM-HG1 under the irradiation of different X-ray dose; (I) the X-ray dose dependent phosphorescence emission spectrum of crystalline LIFM-HG1 at room temperature.

F I G R E 4 (
A and B) The phosphorescent non-radiative transition rate (k nr P ) and intersystem crossing rate (k isc ) of crystalline guest (G) and LIFM-HG1 at room temperature (RT) and 77 K. (C and D) The phosphorescence lifetime and total quantum yield ratios of crystalline LIFM-HG1 and G at room temperature (RT) and 77 K. (E) Two-photon emission and absorption cross sections (σ) of crystalline LIFM-HG1.(F) The phosphorescence intensity and lifetime ratios of crystalline LIFM-HG1 and G under X-ray excitation at room temperature.(G) The ratios of A P /A F for crystalline LIFM-HG1 under the excitation of single-photon, two-photon and X-ray, wherein the A P and A F describe the integral area of room temperature phosphorescence and fluorescence region, respectively.

F I G U R E 5
LPL of crystalline LIFM-HG1 in different time intervals after excitation at by single-photon (λ ex = 310 nm) (A) and twophoton (λ ex = 670 nm) (B) at different temperatures.(C) And the LPL photographs of crystalline LIFM-HG1 after excited by X-ray (power: 9.6 W; dose rate: 982.47 mGy/s) at room temperature.(time scale: s).
This work was supported by NSFC (grant numbers: 22171291, 92261114, 21821003, and 21890380), NKRD Program of China (grant number: 2021YFA1500401), Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (grant number: 2017BT01C161), and FRF for the Central Universities.The authors thank the Tianhe platforms at the National Supercomputer Centers in Guangzhou for their technical support and generous allocation of times.F U N D I N G I N F O R M AT I O N NSFC, Grant Numbers: 22171291, 92261114, 21821003, and 21890380; NKRD Program of China, Grant Number: 2021YFA1500401; Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program, Grant Number: 2017BT01C161; FRF for the Central UniversitiesC O N F L I C T O F I N T E R E S T S TAT E M E N TThe authors declare no conflict of interests.D ATA AVA I L A B I L I T Y S TAT E M E N TThe data that support the findings of this study are available from the corresponding author upon reasonable request.O R C I DQiang-Sheng Zhang https://orcid.org/0000-0001-9728-0296Xiao-Dong Zhang https://orcid.org/0000-0003-3150-2284Jia-Yi Zhuang https://orcid.org/0000-0002-5228-2287Mei Pan https://orcid.org/0000-0002-8979-7305RE F E R E N C E S