Highly reversible supramolecular photo-switching system with macrocycle-mediated photo-induced emission enhancement, preparation and application in targeted cell imaging and anti-counterfeiting

Abstract

Highly reversible supramolecular photoswitching system with macrocycle-mediated photoinduced emission enhancement, preparation and application in targeted cell imaging and anti-counterfeiting. A supramolecular photoswitching system self-assembly with photo-induced crystallization, emission enhancement and assembly-activated emission enhancement photoactivation mechanism was developed using supramolecular assembly, and further assembled with HACD. Due to the reversible photoisomerization characteristics and efficient AIE characteristics of DAE units, the luminescence of DAE-CO can be reversibly turned on / off. Under the stimulation of light, DAE-CO tends to self-aggregate into larger aggregates with emission enhancement characteristics, and this enhancement can be gradually increased by alternating irradiation in several UV / NIR cycles. Like DAE-CO, and both have the characteristics of luminescence adjustment and photo-induced aggregation emission enhancement. In addition, we have applied these supramolecular luminescent materials to information encryption and targeted, light-controlled tumor cell imaging.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of nano-supramolecular light-controlled cell imaging and anti-counterfeiting, in particular, a macrocycle-mediated light-induced emission enhancement high-reversible supramolecular light switch system. BACKGROUND

[0002] Organic luminescent materials with color modulation and fluorescence switching properties controlled by physical or chemical stimuli are very important in basic research and our daily life due to their wide applications in data storage devices, biological markers, sensing, optical memories, etc. Compared with inorganic fluorophores that are excited to higher vibrational and rotational energy states, organic fluorophores based on π-structure extension have many advantages such as rich colors, good tunability, and relatively controllable length. However, many organic skeletons with large π-conjugated frameworks and planar structures often exhibit weak fluorescence in high concentrations and in solid state, resulting in an aggregation-caused quenching (ACQ) effect. Fortunately, since 2001, Tang Benzhong's team has developed an important aggregation-induced emission (AIE) photophysical and chemical platform. The mechanism of the high-efficiency luminescence of AIE materials is the restriction of intramolecular motion (RIM), including the restriction of intramolecular vibration (RIV) and the restriction of intramolecular rotation (RIRs). So far, several stimuli-responsive AIE materials have been prepared. Light as a powerful non-contact stimulus does not produce waste, has high precision and remote control characteristics, and is one of the ideal external stimuli. Therefore, it has attracted great interest to construct an AIE-based light control platform to regulate physical and chemical behaviors. Various methods have been successfully used to realize the construction of light-activated fluorophores with AIE characteristics, although current AIE materials with light response can be constructed by (i) reversible photochromic reactions, (ii) irreversible photochemical reactions, (iii) active oxygen-mediated light activation, etc., but they are all based on the change of photochemical behavior of substances. It is very few to develop a class of AIE materials whose luminescence behavior is regulated only by photophysical behavior. In addition, AIE molecules in single-molecule dispersed state can only exhibit weak luminescence function, inspired by the luminescence mechanism of AIEgens, other methods to prevent intramolecular motion have been developed, such as preparing two-dimensional (2D) metal nanoparticle platforms, forming J-aggregates and supramolecular assemblies can effectively supplement AIE materials.

[0003] From the perspective of structure, cucurbit[n]uril (CB[n])s with high binding affinity to various cationic or neutral guests through hydrophobic and electrostatic interactions can be expected to achieve various luminescent emissions by providing macrocycle confinement, suppressing triplet-state quenching, and limiting the molecular motion of encapsulated non-luminescent dyes. Therefore, it is of great significance to develop new light-controllable CB[n]-mediated supramolecular assemblies with light-induced aggregation-induced emission enhancement capability, but to the best of our knowledge, few reports have been made so far. Hyaluronic acid cyclodextrin, as a kind of excellent tumor cell targeting agent, can be combined with tumor cell specificity. Then it enters the tumor cells in the form of endocytosis. Diarylethene molecules, as photoisomerization molecules, convert between two isomers under different light, exhibiting different luminescent behaviors, and this luminescent behavior can be effectively regulated by external light. Based on the above discussion, a kind of tumor cell targeting, luminescence adjustable, light-induced aggregation-induced emission supramolecular assembly is developed, which has great potential application in cell imaging and anti-counterfeiting. SUMMARY

[0004] One of the purposes of the present application is to solve the problems of aggregation-induced quenching of organic luminescent materials and the unadjustable luminescent intensity using photophysical behavior;

[0005] The second purpose of the present application is to solve the problems of tumor cell targeting light-controlled imaging and information anti-counterfeiting in daily life, and to realize the problems of visualization of tumor cells and encryption of information.

[0006] The present application uses coumarin functionalized diarylethene derivative DAE-CO, CB[7], hyaluronic acid β-cyclodextrin (HACD) to construct a macrocycle-mediated light enhancement and light switch ternary supramolecular assembly DAE-CO⊂CB[7]2&HACD. The assembly can be used as fluorescent ink for anti-counterfeiting and cell imaging.

[0007] Among various photoresponsive groups for constructing supramolecular systems, diarylethene derivatives (DAE) with high photoisomerization quantum yield, excellent fatigue resistance and easy modification of different functional groups have been successfully applied to data storage, molecular machines, optical switches and chirality transfer controllers. At the same time, coumarin and its derivatives are a kind of widely used supramolecular guest, which can be captured by macrocyclic host such as cyclodextrin and CB[n], and has the characteristics of [2+2] cycloaddition under ultraviolet irradiation above 300 nm and photopolymerization under 254 nm. The characteristic supramolecular assembly DAE-CO⊂CB[7]2 with photoresponsive AIE characteristics constructed in the application has experienced the first stage of fluorescence emission enhancement, and presents the light activation phenomenon under continuous light irradiation through the assembly activation enhancement (AAEE) and photoinduced crystallization fluorescence enhancement (PICEE) mechanisms. This supramolecular aggregate further assembles with HACD to form ternary supramolecular nanoparticles DAE-CO⊂CB[7]2&HACD, realizing the second stage of emission enhancement. In addition, benefiting from the reversible photoisomerization of the DAE unit, the fluorescence of DAE-CO, DAE-CO⊂CB[7]2 and DAE-CO⊂CB[7]2&HACD can be turned on / off by alternating irradiation between 254 nm and >600 nm. Finally, this light-adjustable system is successfully used for targeted cell imaging and information encryption.

[0008] The technical scheme of the present application:

[0009] One of the schemes, the macrocycle-mediated light-induced emission enhancement high reversible supramolecular optical switch system includes three AIE materials of DAE-CO, DAE-CO⊂CB[7]2 and DAE-CO⊂CB[7]2&HACD, and the application also provides a preparation method of the three AIE materials.

[0010] In the optical switch system DAE-CO⊂CB[7]2&HACD, cucurbituril[7] and hyaluronic acid β-cyclodextrin are used as rotatable restriction molecules of macrocyclic elements, and diarylethene derivatives are used as AIE molecules with light-adjustable ability, and the chemical structural formula of the construction unit is as follows:

[0011]

[0012] The preparation method of the three AIE materials DAE-CO, DAE-CO⊂CB[7]2 and DAE-CO⊂CB[7]2&HACD in the macrocycle-mediated light-induced emission enhancement high reversible supramolecular optical switch system includes the following steps:

[0013] Step 1, preparation of coumarin diarylethene derivative guest DAE-CO;

[0014] Step 2, Preparation of DAE-CO@CB[7]2 and DAE-CO@CB[7]2&HACD solution in host-guest system of macrocycle-mediated photoinduced emission enhancement high reversible supramolecular photo-switching system;

[0015] Step 1, Preparation of coumarin diarylethene derivative guest DAE-CO, the synthetic route is as follows:

[0016]

[0017] The preparation method is as follows:

[0018] 1,2-bis(2-methyl-5-(pyridin-4-yl)thiophen-3-yl)cyclopent-1-ene1 synthesized according to the literature (1 eq, compound 1) and 6-(bromomethyl)-2H-chromen-2-one2 (3 eq, compound 2) were added to DMF (4 mL), and the mixture was stirred at 100-110 ℃ for another 24 hours. Then the reaction mixture was cooled, and an excess of acetone (200 mL) was added to the solution. The precipitate was filtered and washed with petroleum ether and acetone to obtain the desired compound DAE-CO (98% yield).

[0019] Step 2, Preparation of DAE-CO@CB[7]2 and DAE-CO@CB[7]2&HACD solution in host-guest system of macrocycle-mediated photoinduced emission enhancement high reversible supramolecular photo-switching system, the method is:

[0020] (1) 2 equivalents of cucurbituril [7] were added to the mixed aqueous solution of 1 equivalent of coumarin diarylethene derivative guest DAE-CO obtained in step 1. DAE-CO has a relatively large organic skeleton, so its solubility in water is poor, and DMSO needs to be added to increase its solubility. The addition of DMSO will greatly affect the inclusion of coumarin and CB[7], and finally we determine to add 1% DMSO to ensure better solubility while the host and guest have larger bonding capacity. The binary supramolecular system DAE-CO@CB[7]2 solution can be obtained by mechanical stirring, and mechanical stirring is a key step.

[0021] (2) After forming a stable binary supramolecular system, 5 equivalents of HACD solution (calculated according to β-CD) were further added to the solution, and the ternary supramolecular system DAE-CO@CB[7]2&HACD solution was obtained after vigorous stirring.

[0022] The concentration of the diarylethene derivative guest DAE-CO, cucurbituril [7], HACD is 0.01 mM, 0.02 mM, 0.05 mM respectively, and the amount of HACD greatly influences the luminescence behavior of the ternary supramolecular system, so we strictly screened the proportion and determined that the optimal concentration is 0.05 mM.

[0023] Scheme two, application of the supramolecular photoswitching system (DAE-CO, DAE-CO⊂CB[7]2 and DAE-CO⊂CB[7]2&HACD) as anti-counterfeiting materials, the implementation method is:

[0024] The DAE-CO and the binary and ternary supramolecular assembly solutions obtained in step 2 are used as fluorescent ink to write three numbers "5", "0" and "3" on a 96-well plate, and then the numbers are observed under sunlight or a 365 nm portable ultraviolet lamp; subsequently, the color and fluorescence of the numbers are observed under irradiation of 254 nm / >600 nm wavelength light.

[0025] Scheme three, application of the supramolecular photoswitching system (DAE-CO, DAE-CO⊂CB[7]2 and DAE-CO⊂CB[7]2&HACD) as targeted light-controlled tumor cell imaging, the implementation method is:

[0026] The DAE-CO and the binary and ternary supramolecular assembly solutions obtained in step 2, the mitochondrial staining agent Mito-Tracker and the nuclear staining agent DAPI are co-cultured with human lung adenocarcinoma cells (A549 cells), and the imaging position of the constructed monomer and assembly is explored by overlapping with the commercial staining agent; further, the luminescence change of the cells after 254 nm light irradiation is explored.

[0027] The advantages and beneficial effects of the present application are:

[0028] It is a challenging goal to construct smart supramolecular assemblies with tunable light-induced crystallization emission enhancement and macrocycle-mediated luminescence. The present invention describes a photo luminescence-enhanced coumarin-modified diarylethene derivative (DAE-CO) with aggregation-induced emission characteristics, which exhibits reversible switching of the structural interconversion by alternating UV / NIR irradiation. Interestingly, DAE-CO shows a clear photoactivation phenomenon under continuous light irradiation, accompanied by an increase in fluorescence at 432-540 nm. Upon assembly of DAE-CO with cucurbit[7]uril, the resulting DAE-CO⊂CB[7]2 has enhanced fluorescence intensity and longer lifetime compared to DAE-CO alone. The binary supramolecular nanoparticles can also be assembled with β-cyclodextrin-grafted hyaluronic acid (HACD) to form DAE-CO⊂CB[7]2&HACD with even stronger fluorescence. The fluorescence of DAE-CO⊂CB[7]2 and DAE-CO⊂CB[7]2&HACD can also be reversibly quenched and recovered by 254 nm / >600 nm light irradiation, accompanied by self-assembly into larger aggregates and further emission enhancement after light irradiation cycles. In particular, this fluorescence enhancement can gradually increase over several cycles. The present invention explores the potential applications of these light-tunable fluorescence enhancement and switching materials in anti-counterfeiting and targeted tumor cell imaging. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Synthetic route for coumarin diarylethene derivative guest DAE-CO.

[0030] Figure 2 NMR1H spectrum of coumarin diarylethene derivative guest DAE-CO.

[0031] Figure 3 NMR13C spectrum of coumarin diarylethene derivative guest DAE-CO.

[0032] Figure 4 NMR1H spectrum of coumarin diarylethene derivative guest OF-DAE-CO after 254 nm and >600 nm light irradiation. 1 H- 1 H COSY spectrum.

[0033] Figure 5 NMR1H spectrum of coumarin diarylethene derivative guest OF-DAE-CO after 254 nm and >600 nm light irradiation.

[0034] Figure 6 Changes in UV-Vis absorption spectra of coumarin diarylethene derivative guest OF-DAE-CO under 254 nm UV light and >600 nm light irradiation, with the inset showing the cyclic changes in absorption intensity at 690 nm.

[0035] Figure 7 Pictures of the Tyndall effect for OF-DAE-CO, CF-DAE-CO, OF-DAE-CO CB[7]2, CF-DAE-CO CB[7]2, OF-DAE-CO CB[7]2 & HACD, CF-DAE-CO CB[7]2 & HACD.

[0036] Figure 8 Cyclic on-off changes in the absorbance intensity of coumarin diarylethylene derivative guest OF-DAE-CO at 425 nm under 254 nm UV light and >600 nm light irradiation.

[0037] Figure 9 Dynamic light scattering particle size for OF-DAE-CO, CF-DAE-CO, OF-DAE-CO CB[7]2, CF-DAE-CO CB[7]2, OF-DAE-CO CB[7]2 & HACD, CF-DAE-CO CB[7]2 & HACD.

[0038] Figure 10 Transmission electron microscopy pictures for OF-DAE-CO, CF-DAE-CO, OF-DAE-CO CB[7]2, CF-DAE-CO CB[7]2, OF-DAE-CO CB[7]2 & HACD, CF-DAE-CO CB[7]2 & HACD.

[0039] Figure 11 Job plot for coumarin diarylethylene derivative guest DAE-CO with CB[7].

[0040] Figure 12 UV-visible absorption spectra of coumarin diarylethylene derivative guest DAE-CO with CB[7] addition.

[0041] Figure 13 Fitting curve of coumarin diarylethylene derivative guest DAE-CO with CB[7] at 383 nm.

[0042] Figure 14 H NMR spectra of DAE-CO with 0 (I), 0.5 (II), 1.0 (III), 1.5 (IV), 2.0 (V), 2.5 (VI), 3.0 (VII), and 4.0 (VIII) equivalents of CB[7]. 1 H NMR spectra.

[0043] Figure 15 2D ROESY spectra of DAE-CO CB[7]2 in D2O.

[0044] Figure 16Mass spectrum of DAE-CO@CB[7]2.

[0045] Figure 17 Mass spectrum of DAE-CO with 0 (I), 1.0 (II), 1.5 (III), 2.0 (IV), 2.5 (V), 3.0 (VI), and 4.0 (VIII) equivalents of β-CD. 1 H NMR spectra.

[0046] Figure 18 2D ROESY spectra of DAE-CO@CB[7]2&β-CD in D2O.

[0047] Figure 19 Mass spectrum of DAE-CO&β-CD.

[0048] Figure 20 Cycling of DAE-CO@CB[7]2 at 540 nm under 254 nm UV light and >600 nm light irradiation.

[0049] Figure 21 Cycling of DAE-CO@CB[7]2&β-CD at 540 nm under 254 nm UV light and >600 nm light irradiation.

[0050] Figure 22 Imaging pictures of A549 cells after co-staining with OF-DAE-CO, Mito Tracker and DAPI before (top) and after (bottom) 254 nm light irradiation.

[0051] Figure 23 Imaging pictures of A549 cells after co-staining with OF-DAE-CO@CB[7]2, Mito Tracker and DAPI before (top) and after (bottom) 254 nm light irradiation.

[0052] Figure 24 a) H NMR spectra of OF-DAE-CO (bottom) and its changes under 254 nm (middle) and >600 nm (top) light irradiation. Inset: changes in the structure of DAE-CO. b) UV-Vis spectra changes of OF-DAE-CO under 254 nm UV light irradiation. Inset: photo of changes before and after irradiation. c) Fluorescence emission spectra of OF-DAE-CO in different ratios of H2O / THF. Inset: fluorescence photos of OF-DAE-CO with 90% THF (left) and 10% THF (right) under 365 nm. d) Fluorescence spectra changes of OF-DAE-CO under 254 nm and >600 nm light irradiation. Inset: changes in the emission intensity at 540 nm in 12 irradiation cycles. 1 H NMR spectra. Inset: changes in the structure of DAE-CO. b) UV-Vis spectra changes of OF-DAE-CO under 254 nm UV light irradiation. Inset: photo of changes before and after irradiation. c) Fluorescence emission spectra of OF-DAE-CO in different ratios of H2O / THF. Inset: fluorescence photos of OF-DAE-CO with 90% THF (left) and 10% THF (right) under 365 nm. d) Fluorescence spectra changes of OF-DAE-CO under 254 nm and >600 nm light irradiation. Inset: changes in the emission intensity at 540 nm in 12 irradiation cycles.

[0053] Figure 25 Fluorescence spectra of DAE-CO with 0 (I), 2.5 (II) and 3.0 (III) equivalents of CB[7] and CB[7] (III). 1 H NMR spectra.

[0054] Figure 26 Fluorescence spectra of DAE-CO after addition of 0, 0.5, 1.0, 1.5 and 2.0 equivalents of CB[7], b) DAE-CO CB[7]2 after addition of 0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 equivalents (calculated as b-CD) of HACD, c) Fluorescence lifetime at 540 nm, d) Fluorescence lifetime at 432 nm.

[0055] Figure 27 Changes in the UV-Vis absorption spectra of a) DAE-CO CB[7]2 under 254 nm UV light and >600 nm light, inset is the back-and-forth change in the absorption intensity at 680 nm, b) CF-DAE-CO CB[7]2 & HACD under 254 nm UV light and >600 nm light, inset is the back-and-forth change in the absorption intensity at 680 nm.

[0056] Figure 28 Changes in the fluorescence emission spectra of a) DAE-CO CB[7]2 under 254 nm UV light and >600 nm light, inset is the back-and-forth change in the emission intensity at 425 nm, b) CF-DAE-CO CB[7]2 & HACD under 254 nm UV light and >600 nm light, inset is the back-and-forth change in the emission intensity at 425 nm.

[0057] Figure 29 Encrypted anti-counterfeiting pictures of DAE-CO, DAE-CO CB[7]2, DAE-CO CB[7]2 & HACD under sunlight and UV light.

[0058] Figure 30 Imaging pictures of A549 cells co-stained with OF-DAE-CO CB[7]2 & HACD, MitoTracker and DAPI before (top) and after (bottom) 254 nm light irradiation.

[0059] Figure 31 Schematic diagram of the macrocycle-mediated photoinduced emission enhancement of the highly reversible supramolecular photoswitching system. DETAILED DESCRIPTION

[0060] EXAMPLE:

[0061] A macrocycle-mediated photoinduced emission enhancement high reversible supramolecular photo-switching system, which comprises three AIE materials of DAE-CO, DAE-CO⊂CB[7]2 and DAE-CO⊂CB[7]2&HACD.

[0062] Wherein cucurbit[7]uril, hyaluronic acid β-cyclodextrin as the construction of macrocyclic molecular rotation restriction, diarylethene derivatives as AIE molecules with light adjustable ability, the chemical structure of its building unit is as follows:

[0063]

[0064] I. Preparation of a macrocycle-mediated photoinduced emission enhancement high reversible supramolecular photo-switching system

[0065] Comprising the following steps:

[0066] Step 1, preparation of coumarin diarylethene derivative guest DAE-CO;

[0067] The synthetic route (see Figure 1 ) is as follows:

[0068]

[0069] The preparation method is:

[0070] The compound 1 (1 eq) and compound 2 (3 eq) synthesized according to the literature were added into DMF (4 mL), and the mixture was stirred at 100℃ for another 24 hours. Then the reaction mixture was cooled, and an excess of acetone (200 mL) was added to the solution. The precipitate was filtered and washed with petroleum ether and acetone to obtain the desired compound DAE-CO (98% yield).

[0071] Figure 2 It is the nuclear magnetic hydrogen spectrum of coumarin diarylethene derivative guest DAE-CO. The figure shows that the structure of DAE-CO is correct.

[0072] Figure 3 It is the nuclear magnetic carbon spectrum of coumarin diarylethene derivative guest DAE-CO. The figure shows that the structure of DAE-CO is correct.

[0073] Figure 4 It is the nuclear magnetic hydrogen spectrum of coumarin diarylethene derivative guest DAE-CO. 1 H- 1 H COSY spectrum. The figure shows that the assignment of DAE-CO in D2O is correct.

[0074] Step 2, Preparation of DAE-CO⊂CB[7]2 and DAE-CO⊂CB[7]2&HACD solution in macrocycle-mediated photoinduced emission enhancement high-reversible supramolecular photo-switchable system, the method is as follows:

[0075] (1) 2 equivalents of cucurbituril[7] were added to the mixed aqueous solution of coumarin diarylethene derivative guest DAE-CO obtained in step 1 (1% DMSO was added at the same time), and the solution of binary supramolecular system DAE-CO⊂CB[7]2 was obtained by mechanical stirring.

[0076] (2) 5 equivalents of HACD solution (calculated according to β-CD) were further added to the solution, and the solution of ternary supramolecular system DAE-CO⊂CB[7]2&HACD was obtained after vigorous stirring.

[0077] Among them, the concentrations of diarylethene derivative guest DAE-CO, cucurbituril[7], and HACD are 0.01 mM, 0.02 mM, and 0.05 mM, respectively.

[0078] II. Application of supramolecular photo-switchable system assembly solution as anti-counterfeiting material in anti-counterfeiting, the implementation method is:

[0079] DAE-CO and the binary and ternary supramolecular assembly solutions obtained in step 2 above were used as fluorescent ink to write the numbers "5", "0", and "3" on a 96-well plate, and the plate was observed under daylight or 365 nm portable ultraviolet light; then the color and fluorescence of the numbers were observed under 254 nm / >600 nm light irradiation.

[0080] III. Application of supramolecular photo-switchable system assembly solution as light-controlled tumor cell targeting imaging agent in imaging, the implementation method is:

[0081] DAE-CO and the binary and ternary supramolecular assembly solutions obtained in step 2 above, mitochondrial stain Mito-Tracker, and nuclear stain DAPI were respectively co-cultured with human lung adenocarcinoma cells (A549 cells), and the imaging position of the constructed monomers and assemblies was explored by overlapping with commercial stains. The change in cell luminescence after 254 nm light irradiation was further explored.

[0082] Figure 5 NMR spectrum of coumarin diarylethene derivative guest OF-DAE-CO after 254 nm and >600 nm light irradiation. It is shown in the figure that with the increase of 254 nm light irradiation time, the open ring state DAE-CO can gradually change into the closed ring state DAE-CO. The ring-closing efficiency of OF-DAE-CO under 254 nm ultraviolet light irradiation can reach 99%, and the equivalent ring-opening can be achieved under >600 nm light irradiation.

[0083] Figure 6 The UV-Vis absorption spectra of coumarin diarylethene derivative guest OF-DAE-CO under 254 nm UV light and >600 nm light irradiation, the inset is the cyclic changes of the absorption intensity at 690 nm. The figure shows that after a 254 nm / >600 nm light cycle, the absorption peak position of the UV-Vis absorption spectrum of OF-DAE-CO does not change, but the intensity of the absorption peak is weakened, and OF-DAE-CO can itself aggregate to form larger aggregates under light irradiation.

[0084] Figure 7 The pictures of OF-DAE-CO, CF-DAE-CO, OF-DAE-CO⊂CB[7]2, CF-DAE-CO⊂CB[7]2, OF-DAE-CO⊂CB[7]2&HACD's Tyndall effect. The figure shows that OF-DAE-CO, OF-DAE-CO⊂CB[7]2, OF-DAE-CO⊂CB[7]2&HACD are converted into CF-DAE-CO, CF-DAE-CO⊂CB[7]2, CF-DAE-CO⊂CB[7]2&HACD respectively and aggregate after light irradiation, producing Tyndall effect.

[0085] Figure 8 The cyclic changes of the absorption intensity of coumarin diarylethene derivative guest OF-DAE-CO at 425 nm under 254 nm UV light and >600 nm light irradiation. The figure shows that after multiple cycles of 254 nm or >600 nm light irradiation, the UV-Vis absorption intensity of OF-DAE-CO at 425 nm gradually increases.

[0086] Figure 9 The dynamic light scattering particle size of OF-DAE-CO, CF-DAE-CO, OF-DAE-CO⊂CB[7]2, CF-DAE-CO⊂CB[7]2, OF-DAE-CO⊂CB[7]2&HACD. The figure shows that OF-DAE-CO (a), OF-DAE-CO⊂CB[7]2 (c), OF-DAE-CO⊂CB[7]2&HACD (e) are converted into CF-DAE-CO (b), CF-DAE-CO⊂CB[7]2 (d), CF-DAE-CO⊂CB[7]2&HACD (f) respectively and aggregate after light irradiation, and the particle size is larger respectively.

[0087] Figure 10Transmission electron microscope images of OF-DAE-CO (a), CF-DAE-CO (b), OF-DAE-CO@CB[7]2 (c), CF-DAE-CO@CB[7]2 (d), OF-DAE-CO@CB[7]2&HACD (e), CF-DAE-CO@CB[7]2&HACD (f). The images show that OF-DAE-CO, OF-DAE-CO@CB[7]2, OF-DAE-CO@CB[7]2&HACD are converted into CF-DAE-CO, CF-DAE-CO@CB[7]2, CF-DAE-CO@CB[7]2&HACD and aggregate after light irradiation, and the original small-sized nanoparticles are transformed into larger-sized nanoparticles.

[0088] Figure 11 Job plot of coumarin diarylethene derivative guest DAE-CO and CB[7]. The images show that DAE-CO and CB[7] are bonded in a 2:1 ratio in solution.

[0089] Figure 12 UV-Vis absorption spectra of coumarin diarylethene derivative guest DAE-CO with the addition of CB[7]. The images show that the absorption intensity of DAE-CO at 250-450 nm gradually decreases with the addition of CB[7], and DAE-CO and CB[7] effectively assemble.

[0090] Figure 13 Fitting curve of coumarin diarylethene derivative guest DAE-CO at 383 nm and CB[7]. The images show that the bonding constant of DAE-CO and CB[7] can reach 3.49 × 10 9 M –2 .

[0091] Figure 14 H NMR spectra of DAE-CO with 0 (I), 0.5 (II), 1.0 (III), 1.5 (IV), 2.0 (V), 2.5 (VI), 3.0 (VII), and 4.0 (VIII) equivalents of CB[7]. The images show that as the amount of CB[7] increases, the peaks attributed to DAE-CO gradually disappear, and when it is greater than or equal to 2 eq, the position of the DAE-CO peak does not change. This also indirectly proves the 2:1 bonding ratio of host-guest bonds. 1 H NMR spectra of DAE-CO with 0 (I), 0.5 (II), 1.0 (III), 1.5 (IV), 2.0 (V), 2.5 (VI), 3.0 (VII), and 4.0 (VIII) equivalents of CB[7]. The images show that as the amount of CB[7] increases, the peaks attributed to DAE-CO gradually disappear, and when it is greater than or equal to 2 eq, the position of the DAE-CO peak does not change. This also indirectly proves the 2:1 bonding ratio of host-guest bonds.

[0092] Figure 15 2D ROESY spectra of DAE-CO@CB[7]2 in D2O. The images show that there is a spatial H-H correlation between DAE-CO and CB[7].

[0093] Figure 16is the mass spectrum of DAE-CO CB[7]2. The figure shows that there is a strong peak at the position of mass-to-charge ratio 1528.94, which is attributed to [DAE-CO CB[7]2-2Br 2+ , further proving the formation of DAE-CO CB[7]2.

[0094] Figure 17 is the UV-Vis spectrum of DAE-CO CB[7]2 with 0 (I), 1.0 (II), 1.5 (III), 2.0 (IV), 2.5 (V), 3.0 (VI), and 4.0 (VIII) equivalents of β-CD 1 H NMR spectrum. The figure shows that the chemical shifts of all protons attributed to coumarin and pyridine groups remain essentially unchanged, while there is a significant high-field shift of the remaining protons (H9-H12), indicating that β-CD is associated with the cyclopentane moiety in DAE-CO that is not encapsulated by CB[7].

[0095] Figure 18 is the 2D ROESY spectrum of DAE-CO CB[7]2 & β-CD in D2O. The figure shows that there is a significant NOE cross peak between DAE-CO CB[7]2 and β-CD, providing more reliable evidence of assembly.

[0096] Figure 19 is the mass spectrum of DAE-CO & β-CD. The figure shows that there is a strong peak at the position of mass-to-charge ratio 933.28, which is attributed to [DAE-CO & β-CD − 2Br − ] 2+ , further proving that DAE-CO can be bonded with β-CD.

[0097] Figure 20 is the cyclic variation of the absorbance intensity at 540 nm of DAE-CO CB[7]2 under 254 nm UV light and >600 nm light irradiation. The figure shows that after one cycle of 254 nm / >600 nm light irradiation, the fluorescence of DAE-CO CB[7]2 at 540 nm not only recovers but also gradually increases in intensity.

[0098] Figure 21 is the cyclic variation of the absorbance intensity at 540 nm of DAE-CO CB[7]2 & β-CD under 254 nm UV light and >600 nm light irradiation. The figure shows that after one cycle of 254 nm / >600 nm light irradiation, the fluorescence of DAE-CO CB[7]2 & HACD at 540 nm not only recovers but also gradually increases in intensity.

[0099] Figure 22Images of A549 cells co-stained with OF-DAE-CO, Mito Tracker, and DAPI before (top) irradiation with 254 nm light (bottom). The images show that OF-DAE-CO can target tumor cell nuclei for imaging, and the imaging can be modulated by 254 nm light. However, the imaging intensity is relatively low.

[0100] Figure 23 Images of A549 cells after co-staining with OF–DAE–CO⊂CB[7]2, MitoTracker, and DAPI before (top) irradiation with 254 nm light (bottom). The images show that OF–DAE–CO⊂CB[7]2 can target tumor cell nuclei for imaging, and the imaging can be modulated by 254 nm light. The imaging intensity is higher than that of OF–DAE–CO.

[0101] Figure 24 a) OF–DAE–CO (bottom) and its illumination at 254 nm (middle) and >600 nm (top). 1 b) Changes in H NMR spectrum. Inset: Changes in the DAE-CO structure. c) Changes in the UV-Vis spectrum of OF-DAE-CO under 254 nm UV irradiation. Inset: Photographs showing the changes before and after irradiation. d) Fluorescence emission spectra of OF-DAE-CO in different H₂O / THF ratios. Inset: Fluorescence photographs of OF-DAE-CO with 90% THF content (left) and 10% THF content (right) at 365 nm. e) Changes in the fluorescence spectrum of OF-DAE-CO under 254 nm and >600 nm irradiation. Inset: Emission intensity changes at 540 nm over 12 irradiation cycles. The figure shows that under 254 nm / >600 nm irradiation, [the desired structure / structure] can be achieved. 1 The complete transformation of the ¹H NMR spectrum indicates that the DAE core possesses excellent photochromic ability and that the coumarin component is stable under the applied light. The UV-Vis spectrum of OF–DAE–CO shows three maximum absorption points at 273, 329, and 386 nm. When the OF–DAE–CO solution is irradiated with 254 nm light, the peak intensity at 273 nm gradually increases, while the peak intensities at 329 and 386 nm decrease. Simultaneously, a new peak at 690 nm appears and increases, which can be attributed to CF–DAE–CO. Furthermore, three isoabsorption points appear at 316, 431, and 502 nm during the transformation. The color of the solution changes from colorless to green. When THF (R) in the solution... THF As the proportion of R gradually increases, the fluorescence emission band of CF–DAE–CO undergoes a significant blue shift, and the fluorescence intensity increases. THF When R = 0%, it exhibits weak orange-yellow fluorescence, but when R...THF = 90%, strong orange fluorescence appeared, indicating the AIE feature of DAE-CO.

[0102] When OF-DAE-CO was excited at 380 nm, the fluorescence spectrum showed two emission peaks centered at 540 and 425 nm. Under irradiation at 254 nm, the fluorescence intensity decreased at 540 nm and increased at 425 nm. When illuminated with >600 nm light, the fluorescence recovered with the increase of intensity. It is worth noting that the fluorescence intensity of DAE-CO gradually increased under light stimulation at different wavelengths (254 nm / >600 nm), and the formation of light-mediated more compact molecular packing may be the key reason for the fluorescence enhancement.

[0103] Figure 25 For CB[7] (I), 2.5 (II) and CB[7] (III) containing 0, 2.5, 5, 10 and 15 equivalents of DAE-CO 1 H NMR spectra. The chemical shifts of protons located on the coumarin group (H1, H3, H4 and H5), methylene (H6) and pyridine group (H7) showed shifts to high field (Δδ of H1, H3, H4, H5, H6 and H7 were 0.64, 1.02, 1.07, 1.02, 0.53 and 0.44 ppm, respectively). The other protons (H8) on pyridine, thiophene ring (H9), cyclopentenyl (H10 and H11) and methyl (H12) showed shifts to low field (Δδ = 0.04−0.40 ppm).

[0104] Figure 26(a) Fluorescence spectra of DAE-CO after addition of 0, 0.5, 1.0, 1.5 and 2.0 equivalents of CB[7], b) Fluorescence spectra of DAE-CO CB[7]2 after addition of 0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 equivalents (calculated as β-CD) of HACD, c) Fluorescence lifetime at 540 nm, d) Fluorescence lifetime at 432 nm. The figure shows that the fluorescence intensity of DAE-CO at 432 and 540 nm increases by 2.3 and 4.6 folds, respectively, with the gradual addition of CB[7] due to the conformational restriction caused by host-guest inclusion. The fluorescence of DAE-CO becomes strong orange after the addition of 2 equivalents of CB[7]. The fluorescence intensity of DAE-CO CB[7]2 increases by 1.5 folds upon the addition of 5.0 equivalents of HACD to the solution of DAE-CO CB[7]2. Further increase in the amount of HACD weakens the fluorescence emission intensity. The fluorescence lifetime of DAE-CO CB[7]2 & HACD at 540 nm is measured to be 1458 ps, which is significantly higher than that of DAE-CO CB[7]2 and DAE-CO (1278, 106 ps). Of course, the fluorescence lifetime at 432 nm is consistent with that at 540 nm.

[0105] Figure 27 a) Changes in the UV-Vis absorption spectra of DAE-CO CB[7]2 under 254 nm UV light and >600 nm light irradiation, the inset is the cyclic changes in the absorption intensity at 680 nm, b) Changes in the UV-Vis absorption spectra of CF-DAE-CO CB[7]2 & HACD under 254 nm UV light and >600 nm light irradiation, the inset is the cyclic changes in the absorption intensity at 680 nm. The figure shows that the absorption intensity at 331 nm, 380 nm decreases and the absorption intensity at 273 nm increases when OF-DAE-CO CB[7]2 and CF-DAE-CO CB[7]2 & HACD are irradiated with 254 nm UV light. At the same time, a new absorption peak appears at 680 nm, and three isosbestic points (317 nm, 427 nm and 497 nm) appear, indicating the formation of supramolecular assemblies (CF-DAE-CO CB[7]2 and CF-DAE-CO CB[7]2 & HACD). When the assemblies are irradiated with >600 nm light, the UV-Vis absorption spectrum cannot be restored to its original level, but the position of the absorption peak remains unchanged and the absorption intensity decreases. With the increase in the number of light irradiation cycles, the absorption intensity at 680 nm becomes weaker and weaker.

[0106] Figure 28a) Changes in the fluorescence emission spectra of DAE-CO@CB[7]2 under 254 nm UV light and >600 nm light, inset is the on-off-on changes in the emission intensity at 425 nm, b) Changes in the fluorescence emission spectra of OF-DAE-CO@CB[7]2&HACD under 254 nm UV light and >600 nm light, inset is the on-off-on changes in the emission intensity at 425 nm. It is shown in the figure that after 254 nm UV light irradiation, the peak intensity of OF-DAE-CO@CB[7]2 and OF-DAE-CO@CB[7]2&HACD observed at 540 nm gradually decreased, with quenching efficiencies of 67% and 70%, respectively. Subsequently, the quenched fluorescence emission recovered and became stronger than its initial state when the process was repeated several times between UV and NIR light irradiation, the emission peak at 425 nm increased in intensity.

[0107] Figure 29 Encrypted anti-counterfeiting pictures of DAE-CO, DAE-CO@CB[7]2, DAE-CO@CB[7]2&HACD under sunlight and UV light. It is shown in the figure that three numbers "5", "0", "3" were drawn in 96-well plates with DAE-CO, DAE-CO@CB[7]2 and DAE-CO@CB[7]2&HACD, respectively. Since the solution is colorless, these numbers are hidden under sunlight. When the 96-well plate was placed under a 365 nm UV light, orange fluorescent numbers "503" appeared and the light intensity gradually increased from "5" to "0" and then to "3". After the 96-well plate was exposed to 254 nm UV light, the hidden information could be seen by the naked eye, but no fluorescence was displayed under 365 nm. Interestingly, the information of UV irradiation could be completely recovered after irradiation with near-infrared light (>600 nm) and could be repeated many times.

[0108] Figure 30 Imaging pictures of A549 cells after co-staining with OF-DAE-CO@CB[7]2&HACD, MitoTracker and DAPI before (top) and after (bottom) 254 nm light irradiation. It is shown in the figure that OF-DAE-CO@CB[7]2&HACD can target the nucleus of tumor cells for imaging, and the imaging can be regulated by 254 nm light. The intensity of the imaging is high.

[0109] Figure 31Schematic diagram of high reversible supramolecular photo-switching system for macrocycle-mediated photo-induced emission enhancement, a) indicates that the fluorescence intensity becomes larger after forming binary assembly, the fluorescence intensity is further enhanced after assembling into ternary assembly, and the luminescence behavior at 540 nm can be adjusted; photo-induced aggregation-induced emission enhancement behavior of DAE-CO, DAE-CO⊂CB[7]2, DAE-CO⊂CB[7]2&HACD.

[0110] Table 1 is the fluorescence lifetime at 540 nm.

[0111] Table 1

[0112]

[0113] The table indicates that the fluorescence lifetime at 540 nm gradually becomes longer after assembling into binary and ternary assemblies, and the fluorescence lifetime of OF-DAE-CO⊂CB[7]2&HACD is weakened after 254 nm light irradiation, but becomes longer after >600 nm irradiation.

[0114] Table 2 is the fluorescence lifetime at 432 nm.

[0115] Table 2

[0116]

[0117] The table indicates that the fluorescence lifetime at 432 nm gradually becomes longer after assembling into binary and ternary assemblies, and the fluorescence lifetime of OF-DAE-CO⊂CB[7]2&HACD is enhanced after 254 nm light irradiation, and further becomes longer after >600 nm irradiation.

Claims

1. A method for preparing a macrocyclic-mediated photoemission-enhanced reversible supramolecular optical switch system, wherein the supramolecular optical switch system comprises three AIE materials: DAE–CO, DAE–CO⊂CB[7]2, and DAE–CO⊂CB[7]2&HACD, ​​wherein cucurbituril[7] and hyaluronic acid β-cyclodextrin serve as macrocyclic building blocks to restrict molecular rotation, and diarylene ethylene derivatives serve as AIE molecules with phototunable capabilities. The chemical structural formulas of its building blocks are as follows: The preparation method includes the following steps: Step 1: Preparation of DAE–CO, a coumarin diarylethylene derivative guest; Step 2: Preparation of DAE–CO⊂CB[7]2 and DAE–CO⊂CB[7]2 & HACD solutions in the macrocyclic-mediated photoemission-enhanced reversible supramolecular photoswitching system.

2. The method for preparing a macrocyclic-mediated photoemission-enhanced reversible supramolecular optical switch system as described in claim 1, characterized in that, In step 1, the preparation of the coumarin diarylethylene derivative guest DAE–CO, The synthesis route is as follows: The preparation method is as follows: 1,2-bis(2-methyl-5-(4-pyridyl)thiophene-cyclopent-1-ene and 2-(bromomethyl)-2H-chromen-2-one synthesized according to the literature were added to DMF in an equivalent ratio of 1:3, and the mixture was stirred at 100–110 °C for 24 hours. The reaction mixture was then cooled, and excess acetone was added to the solution. The precipitate was filtered and washed with petroleum ether and acetone to give the desired compound DAE–CO in 98% yield.

3. The method for preparing a macrocyclic-mediated photoemission-enhanced reversible supramolecular optical switch system as described in claim 1, characterized in that, The preparation methods for the binary and ternary supramolecular assembly solutions in the supramolecular photoswitching system described in step 2 are as follows: Two equivalents of cucurbituril[7] were added to a mixed aqueous solution of one equivalent of DAE–CO obtained in step 1, and the solution of the binary supramolecular system DAE–CO⊂CB[7]2 was obtained by mechanical stirring. Further, 5 equivalents of HACD solution were added to the solution, and after vigorous stirring, a solution of the ternary supramolecular system DAE–CO⊂CB[7]2&HACD was obtained. The optimal concentrations of the diaryl ethylene derivative guest DAE–CO, cucurbituril[7], and HACD were 0.01 mM, 0.02 mM, and 0.05 mM, respectively.

4. The application of the macrocyclic photoemission-enhanced reversible supramolecular optical switch system DAE–CO, DAE–CO⊂CB[7]2 and DAE–CO⊂CB[7]2&HACD prepared by the method of claim 1 as an anti-counterfeiting material.

5. The application as described in claim 4, characterized in that, The method of using the application is as follows: the solution of DAE-CO prepared by the method of claim 1, the solution of the binary supramolecular system DAE-CO⊂CB[7]2, and the solution of the ternary supramolecular system assembly DAE-CO⊂CB[7]2&HACD are used as fluorescent inks. The three numbers "5", "0" and "3" are written on a 96-well plate respectively, and observed under sunlight or a 365 nm handheld ultraviolet lamp. Then, the color and fluorescence changes of "5", "0" and "3" are observed by irradiation with light of wavelengths of 254 nm and >600 nm.

6. The application of a macrocyclic-mediated photoemission-enhanced reversible supramolecular optical switch system prepared by the method of claim 1 as a light-controlled tumor cell targeting imaging agent.

7. The application as described in claim 6, characterized in that, The method of using the application is as follows: DAE-CO solution prepared by the method described in claim 1, solution of binary supramolecular system DAE-CO⊂CB[7]2, solution of ternary supramolecular system assembly DAE-CO⊂CB[7]2&HACD solution, mitochondrial staining agent Mito-Tracker and nuclear staining agent DAPI are co-cultured with human lung adenocarcinoma cells. The imaging position of the constructed monomers and assemblies is explored by overlapping with commercial staining agents. The changes in cell luminescence after 254 nm light irradiation are further explored.

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