Endoplasmic reticulum targeted self-assembly AIE probe with cascaded ROS / RNS generation performance

Abstract

The invention provides an ER targeting self-assembly AIE probe with cascade ROS / RNS generation performance, and belongs to the technical field of material chemistry, the probe is composed of four units: 1) an AIE photosensitizer OTBS used for generating ROS; 2) deriving peptide FFVLK from beta amyloid protein, and promoting the self-assembly of the peptide to form a nanofiber with a beta-sheet structure; (3) an oligomerization arginine unit RRRR which is used as an NO donor; and 4) ER targeting signal peptide KDEL to endow ER with targeting ability. Based on ER targeting and a self-assembly process, the self-assembly AIE probe is subjected to ER in-situ self-assembly in cells to form nanofibers, ROS / RNS is generated in a cascading manner under illumination, and strong ICD is induced. In addition, the tumor residence time of the photosensitizer is prolonged due to the formation of the nanofibers, and the treatment effect is further improved. The invention provides a new thought for the design of a novel light-operated II-type ICD inducer.

Description

Technical Field

[0001] The present invention belongs to the field of material chemistry, and particularly relates to an endoplasmic reticulum-targeted self-assembled AIE probe with cascaded ROS / RNS generation performance. Background Art

[0002] Photodynamic therapy (PDT) is a minimally invasive technique with high precision and biosafety, and has become an emerging tumor treatment method. Its main principle is to utilize photosensitizers to accumulate locally in tumors and absorb light energy, thereby generating reactive oxygen species (ROS) to kill tumor cells. A large number of studies have confirmed that while killing tumor cells, PDT can induce immunogenic cell death (ICD) in tumor cells, improve tumor immunogenicity, and then activate the body's anti-tumor immunity, which is particularly suitable for the treatment of immunogenic "cold" tumors. Endoplasmic reticulum (ER) stress is the main initiation program for the occurrence of ICD. According to the degree of ICD induction, ICD inducers can be divided into two types, namely type I ICD inducers (non-ER-targeted drugs inducing mild ER stress) and type II ICD inducers (ER-targeted drugs inducing strong ER stress). Compared with the former, the anti-tumor immune activation effect of type II ICD inducers is more powerful and persistent. Therefore, the development of type II ICD inducers is of great significance for promoting the clinical transformation of PDT in tumor treatment.

[0003] When traditional photosensitizers target and aggregate in tumor tissues or cells, due to the aggregation-induced quenching (ACQ) phenomenon, it usually inevitably leads to a decrease in fluorescence intensity and ROS production, thereby weakening their therapeutic effects. In contrast, photosensitizers with aggregation-induced emission (AIE) characteristics will exhibit enhanced fluorescence and ROS yields due to the restriction of intramolecular motion when aggregating at biological targets, so they are more suitable for tumor optical diagnosis and treatment. Encapsulation by nanocarriers is an effective way to improve the therapeutic potential of AIE photosensitizers. However, inside the nanocarriers, the aggregation of AIE molecules often shows a disordered state, which to a certain extent limits the biological therapeutic efficacy of AIE molecules.

[0004] As the main effector molecule of PDT in inhibiting tumor activity, ROS still faces severe challenges in the treatment of solid tumors. On the one hand, the hypoxic environment in solid tumors leads to most photosensitizers being unable to effectively generate ROS to kill tumor cells; on the other hand, the highly reducing environment in tumors can eliminate ROS, further inhibiting the efficacy of PDT. In addition, the rapid metabolism of photosensitizers at the tumor site further limits the therapeutic effect of PDT to a certain extent. Summary of the Invention

[0005] In order to solve the problems of hypoxia and highly reducing environment, poor aggregation of AIE molecules, weak activation degree of ICD, and fast metabolism faced by existing PDT-based ICD therapies in solid tumor immunotherapy, the present invention proposes an ER-targeted self-assembled AIE probe with cascaded ROS / Reactive nitrogen species (RNS) generation performance. Through the ER-targeting and self-assembly processes, it self-assembles into nanofibers in situ at the ER of tumor cells, and cascades to generate ROS / RNS under light irradiation, inducing strong ICD. In addition, the formation of nanofibers effectively increases the tumor retention time of the photosensitizer, further improving the therapeutic effect.

[0006] The present invention provides an ER-targeted self-assembled AIE probe with cascaded ROS / RNS generation performance. The AIE probe includes four functional units: OTBS, FFVLK, RRRR, and KDEL, and its molecular structure is as follows:

[0007]

[0008] This molecule consists of four functional units:

[0009] 1) The AIE photosensitizer molecule 4-(4-(2-(7-(4-(bis(4-methoxyphenyl)amino)phenyl)benzo[c][1,2,5]thiadiazol-4-yl)-1-cyanoethenyl)phenyl)-1-(hept-6-yn-1-yl)pyridin-1-ium cation (OTBS), which is used to provide a hydrophobic end and generate ROS;

[0010] 2) The amyloid-β (Aβ)-derived amino acid sequence FFVLK, which can promote the self-assembly of peptides to form nanofibers with a β-sheet protein secondary structure;

[0011] 3) The oligolysine unit RRRR, which serves as a NO generation donor under light irradiation;

[0012] 4) The ER-targeting signal peptide KDEL, which endows the probe with ER-targeting ability.

[0013] The synthesis steps are as follows:

[0014] Step 1. Synthesis of OTBS:

[0015] (1) Dissolve 4-bromo-N,N-bis(4-methoxyphenyl)aniline, bis(pinacolato)diboron, and tetrakis(triphenylphosphine)palladium in a tetrahydrofuran (THF) solution. Subsequently, introduce an aqueous solution of potassium carbonate (K2CO3) into the reaction vessel. Under a nitrogen atmosphere, heat the mixture to 80 °C and reflux for 8 hours. After the reaction is completed, extract the mixture with a saturated sodium chloride (NaCl) aqueous solution and collect the organic phase. Obtain the crude product by rotary evaporation. After purification, 4-borate-4',4'-dimethoxytriphenylamine is obtained;

[0016] The molar ratio of 4-bromo-N,N-bis(4-methoxyphenyl)aniline, bis(pinacolato)diboron, and tetrakis(triphenylphosphine)palladium is (0.5 - 1):1:0.01, preferably 0.75:1:0.01;

[0017] (2) Dissolve 4-borate-4',4'-dimethoxytriphenylamine, 7-bromo-benzo[c][1,2,5]thiadiazole-4-carbaldehyde, and tetrakis(triphenylphosphine)palladium in a THF solution. Subsequently, introduce an aqueous solution of K2CO3 into the reaction vessel. Under a nitrogen atmosphere, heat the mixture to 80 °C and reflux for 8 hours. After the reaction is completed, extract the mixture with a saturated NaCl aqueous solution and collect the organic phase. Obtain the crude product by rotary evaporation. After purification, 7-(4-(bis(4-methoxyphenyl)amino)benzo)benzo[c][1,2,5]thiadiazole-4-carbaldehyde is obtained;

[0018] The molar ratio of 4-borate-4',4'-dimethoxytriphenylamine, 7-bromo-benzo[c][1,2,5]thiadiazole-4-carbaldehyde, and tetrakis(triphenylphosphine)palladium is (0.5 - 1):1:0.01, preferably 0.80:1:0.01;

[0019] (3) Dissolve 7-(4-(bis(4-methoxyphenyl)amino)benzo)benzo[c][1,2,5]thiadiazole-4-carbaldehyde, 2-(4-(pyridin-4-yl)phenyl)acetonitrile, and sodium ethoxide in absolute ethanol (EtOH), and stir at room temperature for 24 hours under a nitrogen atmosphere. After the reaction is completed, obtain the crude product by rotary evaporation and obtain 3-(7-(4-(bis(4-methoxyphenyl)amino)phenyl)benzo[c][1,2,5]thiadiazole-4-yl)-2-(4-(pyridin-4-yl)phenyl)acrylonitrile by purification;

[0020] The molar ratio of 7-(4-(bis(4-methoxyphenyl)amino)benzo)benzo[c][1,2,5]thiadiazole-4-carbaldehyde, 2-(4-(pyridin-4-yl)phenyl)acetonitrile, and sodium ethoxide is 1:(1 - 1.5):0.1, preferably 1:1.2:0.1;

[0021] (4) Dissolve 3-(7-(4-(bis(4-methoxyphenyl)amino)phenyl)benzo[c][1,2,5]thiadiazol-4-yl)-2-(4-(pyridin-4-yl)phenyl)acrylonitrile and 6-iodo-1-hexyne in N,N-dimethylformamide (DMF) solution, heat to 80 °C and reflux for 16 h under a nitrogen atmosphere. After the reaction is completed, obtain the crude product by rotary evaporation. After purification, dissolve the product in methanol (MeOH) solution. Add an equal volume of aqueous potassium hexafluorophosphate (KPF6) solution, stir at room temperature for 5 h. Wash the resulting mixture three times with dichloromethane (DCM) for extraction, retain the organic phase and perform rotary evaporation to obtain the hexafluorophosphate of OTBS (4-(4-(2-(7-(4-(bis(4-methoxyphenyl)amino)phenyl)benzo[c][1,2,5]thiadiazol-4-yl)-1-cyanoethenyl)phenyl)-1-(hept-6-yn-1-yl)pyridin-1-ium), for use;

[0022] The molar ratio of 3-(7-(4-(bis(4-methoxyphenyl)amino)phenyl)benzo[c][1,2,5]thiadiazol-4-yl)-2-(4-(pyridin-4-yl)phenyl)acrylonitrile to 6-iodo-1-hexyne is (0.5 - 1) : 1, preferably 0.78 : 1.

[0023] Step Two: Synthesis of the polypeptide:

[0024] Synthesize the polypeptide sequentially from the carboxyl terminus (right) to the amino terminus (left) of the polypeptide sequence by solid phase peptide synthesis (SPPS):

[0025] (1) Dissolve dichloride resin in DCM, place it in a solid phase tube for swelling, and after completion, squeeze out the DCM with an ear bulb;

[0026] (2) Weigh Fmoc-protected leucine (Fmoc-Leu-OH) and dissolve it in DMF, add N,N-diisopropylethylamine (DIPEA) to adjust the pH to 8 - 9, add it to the solid phase tube, and react on a shaker at room temperature for 2 h;

[0027] (3) Wash 5 times with DCM, add a blocking solution (mass ratio DCM:MeOH:DIPEA = 17:2:1) for blocking, and after completion, wash 5 times with DCM and DMF respectively;

[0028] (4) Add a DMF solution containing 20% (v / v) piperidine to cleave the Fmoc protecting group, and after completion, wash 5 times with DMF;

[0029] (5) Weigh the second amino acid, i.e., Fmoc-protected glutamic acid (Fmoc-Glu-OH), dissolve it in DMF together with O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), add DIPEA to adjust the pH to 8 - 9, add it into the solid-phase tube, and react on a shaker at room temperature for 2 h;

[0030] (6) Repeat steps (4) and (5), and successively add Fmoc-protected aspartic acid (Fmoc-Asp-OH), Fmoc-protected lysine (Fmoc-Lys-OH), Fmoc-protected arginine (Fmoc-Arg-OH), Fmoc-protected arginine (Fmoc-Arg-OH), Fmoc-protected arginine (Fmoc-Arg-OH), Fmoc-protected arginine (Fmoc-Arg-OH), Fmoc-protected lysine (Fmoc-Lys-OH), Fmoc-protected leucine (Fmoc-Leu-OH), Fmoc-protected valine (Fmoc-Val-OH), Fmoc-protected phenylalanine (Fmoc-Phe-OH), Fmoc-protected phenylalanine (Fmoc-Phe-OH), and azidoacetic acid;

[0031] (7) Add the polypeptide cleavage solution (95% (v / v) trifluoroacetic acid + 2.5% (v / v) trimethylsilane + 2.5% (v / v) water) to cleave the polypeptide, collect the cleavage solution in a round-bottom flask, wash it 3 - 5 times with DCM solution containing 1% (v / v) trifluoroacetic acid, evaporate the collected solution to dryness using a rotary evaporator, add anhydrous ether to the dried substance, precipitate a solid, blow dry the ether, and collect the solid;

[0032] (8) Use high-performance liquid chromatography to purify the product to obtain the polypeptide N3-FFVLKRRRRKDEL, evaporate methanol, and use a freeze dryer to remove water to obtain a solid product for standby.

[0033] The molar ratio of dichlororesin to leucine is 1: (1.5 - 2.5), preferably 1: 2.

[0034] Step 3: Synthesis of OTBS-FR-ER:

[0035] Dissolve the polypeptide N3-FFVLKRRRRKDEL and OTBS in dimethyl sulfoxide (DMSO), and adjust the pH value of the solution to 8 - 9 with DIPEA; add copper sulfate pentahydrate (CuSO4·5H2O) and sodium ascorbate to initiate the click chemical reaction, stir at room temperature in a nitrogen environment for 36 hours, and after the reaction, purify it using HPLC to obtain the product OTBS-FR-ER.

[0036] The molar ratio of polypeptide N3-FFVLKRRRRKDEL to OTBS is (1.5 - 2):1, preferably 1.5:1.

[0037] The present invention also provides the application of the above ER-targeted self-assembled AIE probe with cascade ROS / RNS generation performance in the preparation of optical diagnostic or therapeutic reagents for prostate tumors.

[0038] Beneficial effects

[0039] (1) The self-assembled AIE probe OTBS-FR-ER provided by the present invention can effectively target the ER of tumor cells under the action of the "KDEL" targeting sequence, and self-assemble into nanofibers with a β-sheet protein secondary structure at the ER site under the promotion of the "FFVLK" self-assembly unit, promoting the ordered arrangement of AIE molecules and improving the fluorescence and ROS generation effects of OTBS;

[0040] (2) The AIE molecule OTBS provided by the present invention is an outstanding dual-type photosensitizer, which can generate type I ROS, such as hydroxyl radical (•OH) and superoxide anion (O2 •- ), and type II ROS, such as singlet oxygen ( 1 O2), under photoactivation. Compared with traditional type II photosensitizers, OTBS can, to a certain extent, break through the tumor hypoxia limitation. On this basis, by combining OTBS with oligolysine "RRRR", ROS can be converted into more oxidizing RNS, including nitric oxide (NO) and its oxidation product peroxynitrite anion (ONOO - ), further effectively breaking through the limitations of tumor hypoxia and high reduction environment and improving the PDT efficacy.

[0041] (3) In the present invention, the NO donor oligolysine "RRRR" is covalently linked to the self-assembled AIE probe, which can break through the limitations of short ROS half-life (less than 40 ns) and limited diffusion distance (about 10 nm). Under photoactivation, the ROS generated by OTBS can rapidly oxidize oligolysine, promoting the generation of RNS. Through the combined action of ROS / RNS, ER stress is directly induced, activating strong ICD. Therefore, OTBS-FR-ER is a highly efficient novel light-controlled type II ICD inducer.

[0042] (4) In the present invention, the formation of OTBS-FR-ER nanofibers can effectively prolong the retention time of the photosensitizer in the tumor, further improving the therapeutic effect. Brief description of the drawings

[0043] Figure 1 For the AIE molecule OTBS (20 μM) of the present invention in aqueous solution: (a) UV-Vis absorption spectrum, (b) fluorescence emission spectrum, and (c) AIE property;

[0044] Figure 2 For the AIE molecule OTBS (1 mM) of the present invention under white light irradiation (0.25 W cm -2 ), the ROS generation performance and types were detected by an Electron paramagnetic resonance (EPR) spectrometer; where a is hydroxyl radical (·OH), b is superoxide anion radical (O2 •- ), and c is singlet oxygen ( 1 O2);

[0045] Figure 3 For three AIE probes (20 μM) of the present invention in aqueous solution: (a) UV-Vis absorption spectrum and (b) fluorescence emission spectrum.

[0046] Figure 4 For three AIE probes of the present invention: (a) Transmission electron microscope (TEM) images, (b) Circular dichroism (CD) spectra, and (c) Critical aggregation concentration (CMC) values.

[0047] Figure 5 For three AIE probes (20 μM) of the present invention under white light irradiation (0.25 W cm -2 ): (a) Total ROS, (b) O2 •- , (c) NO, and (d) ONOO - generation curves.

[0048] Figure 6 For three AIE probes of the present invention: (a) OTBS-FR-ER, (b) OTBS-R-ER, and (c) OTBS-FK-ER (20 μM) in RM-1 tumor cells, ER-targeted fluorescence co-localization laser scanning confocal microscopy (CLSM) images.

[0049] Figure 7 For three AIE probes (20 μM) of the present invention in the dark environment and under white light irradiation (0.25 W cm -2 ): (a) Total ROS, (b) NO, and (c) ONOO - detection CLSM images of generation ability.

[0050] Figure 8This is the biological transmission electron microscopy (Bio-TEM) image of the self-assembled AIE probe OTBS-FR-ER (20 μM) of the present invention inducing morphological changes in ER of RM-1 tumor cells under white light irradiation (0.25 W cm -2 )

[0051] Figure 9 This is the result of the cytotoxicity experiment of three AIE probes of the present invention on RM-1 tumor cells under white light irradiation (0.25 W cm -2 )

[0052] Figure 10 This is the result of (a) protein immunoblotting experiment (Western blot) and (b, c) quantitative analysis of apoptosis-related proteins of three AIE probes (20 μM) of the present invention inducing apoptosis in RM-1 tumor cells under white light irradiation (0.25 W cm -2 )

[0053] Figure 11 This is the result of (a) flow cytometry image of calreticulin externalization (ecto-CRT) and (b) ELISA experiment result of high mobility group protein B1 (HMGB1) release of three AIE probes (20 μM) of the present invention inducing ICD in RM-1 tumor cells under white light irradiation (0.25 W cm -2 )

[0054] Figure 12 This is the result of (a) flow cytometry image and (b) quantitative analysis of inducing the maturation of bone marrow-derived dendritic cells (BMDCs) in vitro by activating the immunogenicity of RM-1 cells of three AIE probes (20 μM) of the present invention under white light irradiation (0.25 W cm -2 )

[0055] Figure 13 This is the result of (a) fluorescence imaging and (b) fluorescence quantitative analysis of three AIE probes of the present invention at different observation times after intratumoral injection in mice (100 μL, 100 μM) Detailed implementation mode

[0056] Example 1

[0057] OTBS-FR-ER (endoplasmic reticulum-targeted self-assembled AIE probe with cascade ROS / RNS generation performance)

[0058] 1. Synthesis of OTBS:

[0059]

[0060] (1) Synthesis of compound 1:

[0061] 4-Bromo-N,N-bis(4-methoxyphenyl)aniline (576.41 mg, 1.50 mmol), bis(pinacolato)diboron (507.88 mg, 2.00 mmol) and tetrakis(triphenylphosphine)palladium(0) (23.11 mg, 0.02 mmol) were dissolved in THF solution. Subsequently, 2.00 mol / L aqueous K2CO3 solution (1.10 g, 4.00 mL) was introduced into the reaction vessel. Under a nitrogen atmosphere, the resulting mixture was refluxed at 80 °C for 8 hours. After the reaction was completed, the mixture was cooled to room temperature. Subsequently, the mixture was extracted (washed 3 times with 8.00 mL of saturated NaCl aqueous solution), and the organic phase was collected. The crude product was obtained by rotary evaporation. Finally, 4-boronate-4',4'-dimethoxytriphenylamine (Compound 1) (620.60 mg, 1.44 mmol, 95.94% yield) was obtained after purification.

[0062] 1 H NMR (400 MHz, Chloroform-d) δ 7.60 (d, J = 7.7 Hz, 2H), 7.05 (s,4H), 6.83 (d, J = 9.2 Hz, 6H), 3.80 (s, 6H), 1.32 (s, 12H).

[0063] (2) Synthesis of Compound 2

[0064] 4-Boronate-4',4'-dimethoxytriphenylamine (344.90 mg, 0.80 mmol), 7-bromo-benzo[c][1,2,5]thiadiazole-4-carbaldehyde (243.35 mg, 1.00 mmol) and tetrakis(triphenylphosphine)palladium(0) (11.56 mg, 0.01 mmol) were dissolved in 8.00 mL of THF solution. Subsequently, 2.00 mol / L aqueous K2CO3 solution (1.10 g, 4.00 mL) was introduced into the reaction vessel. Under a nitrogen atmosphere, the resulting mixture was refluxed at 80 °C for 8 hours. After the reaction was completed, the mixture was cooled to room temperature. Subsequently, the mixture was extracted (washed 3 times with 8.00 mL of saturated NaCl aqueous solution), and then the organic phase was collected. Then the crude product was obtained by rotary evaporation. Finally, 7-(4-(bis(4-methoxyphenyl)amino)phenyl)benzo[c][1,2,5]thiadiazole-4-carbaldehyde (Compound 2) (331.66 mg, 0.71 mmol, 88.75% yield) was obtained after purification.

[0065] 11H NMR (400 MHz, Chloroform-d) δ 10.73 (s, 1H), 8.26 (d, J = 7.5 Hz, 1H), 7.90 (d, J = 8.7 Hz, 2H), 7.81 (d, J = 7.4 Hz, 1H), 7.16 (d, J = 8.5 Hz, 4H), 7.03 (d, J = 8.5 Hz, 2H), 6.90 – 6.85 (m, 4H), 3.82 (s, 6H).

[0066] (3) Synthesis of Compound 3

[0067] Dissolve 7-(4-(bis(4-methoxyphenyl)amino)benzo)benzo[c][1,2,5]thiadiazole-4-carbaldehyde (467.13 mg, 1.00 mmol), 2-(4-(pyridin-4-yl)phenyl)acetonitrile (233.08 mg, 1.20 mmol) and sodium ethoxide (6.80 mg, 0.10 mmol) in 8.00 mL of anhydrous EtOH, and stir at room temperature for 24 hours under a nitrogen atmosphere. After the reaction is completed, cool the whole system to room temperature. Subsequently, obtain the crude product by rotary evaporation and purify it to obtain 3-(7-(4-(bis(4-methoxyphenyl)amino)phenyl)benzo[c][1,2,5]thiadiazol-4-yl)-2-(4-(pyridin-4-yl)phenyl)acrylonitrile (Compound 3).

[0068] (4) Synthesis of OTBS

[0069] Dissolve 3-(7-(4-(bis(4-methoxyphenyl)amino)phenyl)benzo[c][1,2,5]thiadiazol-4-yl)-2-(4-(pyridin-4-yl)phenyl)acrylonitrile (500.00 mg, 0.78 mmol) and 6-iodo-1-hexyne (208.04 mg, 1.00 mmol) in 8.00 mL of DMF solution, and reflux at 80 °C for 16 hours under a nitrogen atmosphere. After the reaction is completed, obtain the crude product by rotary evaporation. The separated powder is purified by silica gel column chromatography and then dissolved in 10.00 mL of MeOH solution. Add an equal volume of aqueous KPF6 solution, and stir the mixture on a magnetic stirrer at room temperature for 5 hours. Finally, add 20.00 mL of DCM solution to wash three times for extraction, retain the organic phase and perform rotary evaporation to obtain OTBS (411.15 mg, 0.46 mmol, 58.97% yield).

[0070] 11H NMR (400 MHz, DMSO-d6) δ 9.15 (d, J = 6.4 Hz, 2H), 8.76 – 8.52 (m,4H), 8.28 (d, J = 8.1 Hz, 2H), 8.02 (ddd, J = 32.3, 14.8, 8.4 Hz, 5H), 7.14(d, J = 8.3 Hz, 4H), 6.98 (d, J = 8.6 Hz, 4H), 6.87 (t, J = 7.5 Hz, 2H), 4.63(t, J = 7.2 Hz, 2H), 3.77 (s, 6H), 2.86 (t, J = 2.6 Hz, 1H), 2.26 (td, J =7.1, 2.6 Hz, 2H), 2.04 (p, J = 7.3 Hz, 2H), 1.51 (p, J = 7.2 Hz, 2H). 13 13C NMR(101 MHz, DMSO-d6) δ 156.80, 154.74, 153.87, 152.81, 149.79, 145.34, 139.79,137.31, 135.81, 134.71, 130.77, 129.60, 129.25, 127.88, 127.43, 127.35,126.55, 125.09, 124.33, 118.37, 117.94, 115.58, 84.33, 72.32, 59.99, 55.75,30.38, 24.97, 17.72。

[0071] Figure 1 To detect the ultraviolet absorption, fluorescence emission and AIE properties of the AIE probe OTBS (20 µM). Weigh 1 mg of the AIE molecule OTBS and place it in a centrifuge tube. Dissolve it with DMSO solution to prepare a 2 mM stock solution. Dilute OTBS 1:100 with dd H2O to a 20 µM working solution for subsequent detection. Then use a UV-visible spectrophotometer to measure the ultraviolet-visible absorption spectrum of the AIE probe OTBS, and use a fluorescence spectrometer to detect its fluorescence spectrum. Use a fluorescence spectrometer to measure the photoluminescence properties of the AIE probe OTBS in different aggregation states, that is, place the AIE probe OTBS in DMSO / toluene mixed solutions with different toluene ratios. DMSO is a good solvent for the AIE molecule, and toluene is a poor solvent for the AIE molecule. Measure the fluorescence intensity of the AIE probe OTBS in DMSO / toluene mixed solutions with different toluene ratios.

[0072] As Figure 1 shown in a, b, the maximum absorption wavelength of the AIE probe OTBS is 545 nm, and the maximum fluorescence emission wavelength is 788 nm. As Figure 1 shown in c, the fluorescence emission intensity of the OTBS molecule in DMSO / toluene mixtures with different toluene ratios increases continuously with the increase of the toluene ratio, showing typical AIE characteristics.

[0073] Figure 2 To measure the ROS generation performance and ROS types of the AIE probe OTBS under white light irradiation (0.25 W cm -2 ), the results at 0 minutes and 3 minutes of light irradiation were recorded. The •OH detection procedure is as follows: Take 30 μL of the AIE probe OTBS (1 mM), add 30 μL of the radical scavenger DMPO (100 mM, deionized water as solvent), mix well, then use a capillary to suck a certain amount of the mixture, place it in a quartz tube, and put it into the EPR resonance cavity for the test of •OH. O2 •- The detection procedure is as follows: Take 30 μL of the AIE probe OTBS (1 mM), add 30 μL of the radical scavenger DMPO (100 mM, methanol as solvent), mix well, then use a capillary to suck a certain amount of the mixture, place it in a quartz tube, and put it into the EPR resonance cavity for the test of O2 •- The test. 1 The O2 detection procedure is as follows: Take 30 μL of the AIE probe OTBS (1 mM), add 50 μL of the radical scavenger TEMP (100 mM), mix well, then use a capillary to suck a certain amount of the mixture, put on a quartz tube, and then place it in the EPR sample cavity in a quartz tube and put it into the EPR resonance cavity for the test of 1 O2.

[0074] As Figure 2 shown, the AIE probe OTBS is a potent dual-type photosensitizer that can generate both type I ROS, such as (a) •OH and (b) O2 -2 under white light irradiation (0.25 W cm •- ), and type II ROS, such as (c) 1 O2. At the same time, all three radicals have relatively high yields.

[0075] 2. Synthesis of polypeptides

[0076]

[0077] The polypeptide was synthesized by the SPPS method in sequence from the carboxyl terminus (right) to the amino terminus (left) of the polypeptide sequence.

[0078] (1) Dissolve 0.5 mol of dichloro resin in 15 mL of DCM, add a solid-phase tube, and swell for 10 min. Then, use an ear bulb to squeeze out the DCM.

[0079] (2) Weigh 1 mol of Fmoc-protected leucine (Fmoc-Leu-OH), dissolve it in 15 mL of DMF, add DIPEA to adjust the pH to 8 - 9, add it into the solid-phase tube, and react on a shaker at room temperature for 2 h.

[0080] (3) Wash 5 times with DCM (1 min each time), add a blocking solution (20.00 mL, mass ratio of DCM:MeOH:DIPEA = 17:2:1) and block for 10 - 15 min. After blocking, wash 5 times with DCM and 5 times with DMF respectively (1 min each time).

[0081] (4) Add a DMF solution containing 20% (v / v) piperidine to cleave the Fmoc protecting group for 30 min. After that, wash 5 times with DMF (1 min each time).

[0082] (5) Weigh the second amino acid, i.e., 1 mol of Fmoc-protected glutamic acid (Fmoc-Glu-OH), dissolve it together with 1 mol of HBTU in 15.00 mL of DMF, add DIPEA to adjust the pH to 8 - 9, add it into the solid-phase tube, and react on a shaker at room temperature for 2 h.

[0083] (6) Repeat steps (4) and (5), and successively add Fmoc-protected aspartic acid (Fmoc-Asp-OH), Fmoc-protected lysine (Fmoc-Lys-OH), Fmoc-protected arginine (Fmoc-Arg-OH), Fmoc-protected arginine (Fmoc-Arg-OH), Fmoc-protected arginine (Fmoc-Arg-OH), Fmoc-protected arginine (Fmoc-Arg-OH), Fmoc-protected lysine (Fmoc-Lys-OH), Fmoc-protected leucine (Fmoc-Leu-OH), Fmoc-protected valine (Fmoc-Val-OH), Fmoc-protected phenylalanine (Fmoc-Phe-OH), Fmoc-protected phenylalanine (Fmoc-Phe-OH), and azidoacetic acid.

[0084] (7) Add polypeptide cleavage solution (95% (v / v) trifluoroacetic acid + 2.5% (v / v) trimethylsilane + 2.5% (v / v) water) to cleave the polypeptide for 45 min. Collect the cleavage solution in a round-bottom flask, and wash it 3 - 5 times with DCM solution containing 1% (v / v) trifluoroacetic acid for 1 min each time. After collecting all the solutions, evaporate them to dryness using a rotary evaporator. Add anhydrous ether to the dried substance, and a solid will precipitate. Blow dry the ether and collect the solid;

[0085] (8) Purify the product using high-performance liquid chromatography to obtain N3-FFVLKRRRRKDEL. Evaporate methanol and use a freeze dryer to remove water to obtain a solid product.

[0086] 3. Synthesis of OTBS-FR-ER:

[0087]

[0088] (1) Dissolve N3-FFVLKRRRRKDEL (14.00 mg, 7.40 μmol) and OTBS (3.69 mg, 5.00 μmol) in 2.00 mL of DMSO solution, and adjust the pH value of the solution to 8 - 9 with DIPEA;

[0089] (2) Dissolve CuSO4-5H2O (1.25 mg, 5.00 μmol) and sodium ascorbate (1.98 mg, 10.00 μmol) separately in 100.00 μL of deionized water (dd H2O), and add them to the above DMSO solution in sequence to initiate the click reaction;

[0090] (3) Under a nitrogen atmosphere, stir continuously at room temperature for 36 hours;

[0091] (4) Purify the product using HPLC to obtain OTBS-FR-ER. Evaporate methanol and use a freeze dryer to remove water to obtain a solid product.

[0092] Comparative Example 1

[0093] OTBS-FK-ER (Self-assembled ER-targeting AIE probe that can only generate ROS)

[0094]

[0095] The difference between Comparative Example 1 and Example 1 is as follows: In the synthesis of the polypeptide in Step 2, corresponding amino acids were replaced according to the amino acid sequence. That is, in the 6th step, Fmoc-protected aspartic acid (Fmoc-Asp-OH), Fmoc-protected lysine (Fmoc-Lys-OH), Fmoc-protected lysine (Fmoc-Lys-OH), Fmoc-protected lysine (Fmoc-Lys-OH), Fmoc-protected lysine (Fmoc-Lys-OH), Fmoc-protected lysine (Fmoc-Lys-OH), Fmoc-protected lysine (Fmoc-Lys-OH), Fmoc-protected leucine (Fmoc-Leu-OH), Fmoc-protected valine (Fmoc-Val-OH), Fmoc-protected phenylalanine (Fmoc-Phe-OH), Fmoc-protected phenylalanine (Fmoc-Phe-OH), and azidoacetic acid were added in sequence to form the N3-Phe-Phe-Val-Leu-Lys-Lys-Lys-Lys-Lys-Lys-Asp-Glu-Leu polypeptide sequence, and N3-FFVLKKKKKKDEL was prepared.

[0096] In Step 3, only N3-FFVLKKKKKKDEL was used to replace N3-FFVLKRRRRKDEL, and the remaining steps remained unchanged. Finally, OTBS-FK-ER was prepared.

[0097] Comparative Example 2

[0098] OTBS-R-ER (a cascade ROS / RNS generating ER-targeted self-assembled AIE probe without self-assembly performance)

[0099]

[0100] The difference between Comparative Example 2 and Example 1 is as follows: In the synthesis of the polypeptide in Step 2, corresponding amino acids were replaced according to the amino acid sequence. That is, in the 6th step, Fmoc-protected aspartic acid (Fmoc-Asp-OH), Fmoc-protected lysine (Fmoc-Lys-OH), Fmoc-protected arginine (Fmoc-Arg-OH), Fmoc-protected arginine (Fmoc-Arg-OH), Fmoc-protected arginine (Fmoc-Arg-OH), Fmoc-protected arginine (Fmoc-Arg-OH), and azidoacetic acid were added in sequence to form the N3-Arg-Arg-Arg-Arg-Lys-Asp-Glu-Leu polypeptide sequence, and N3-RRRRKDEL was prepared.

[0101] In step 3, only N3-RRRRKDEL is replaced with N3-FFVLKRRRRKDEL, and the remaining steps remain unchanged. Finally, OTBS-R-ER is prepared.

[0102] Figure 3 The UV-visible absorption spectra and fluorescence emission spectra of three AIE probes (OTBS-R-ER, OTBS-FK-ER, and OTBS-FR-ER) are shown. Weigh 1 mg of AIE probe (OTBS-FR-ER, OTBS-FK-ER, and OTBS-R-ER) and place it in a centrifuge tube. Dissolve it with DMSO solution to prepare a 2 mM stock solution, and dilute the AIE probe 1:100 with dd H2O to a 20 µM working solution for subsequent detection. Then use a UV spectrophotometer to measure the absorption spectrum of the AIE probe and a fluorescence spectrometer to detect its fluorescence spectrum.

[0103] As Figure 3 shown in Fig. a, the three AIE probes show similar UV-visible absorption spectra, indicating that the peptide chain linkage does not change the absorption characteristics of the AIE probe OTBS. However, there are significant differences in the fluorescence emission of the three AIE probes ( Figure 3 Fig. b). Compared with OTBS-R-ER, OTBS-FR-ER and OTBS-FK-ER containing self-assembly units show obvious red-shift and fluorescence enhancement. Based on the aggregation-induced emission enhancement property of AIE molecules, this indicates that the self-assembly unit "FFVLK" promotes the ordered aggregation of OTBS.

[0104] Figure 4To evaluate the self-assembly performance of OTBS-FR-ER, OTBS-FK-ER, and OTBS-R-ER by Transmission electron microscopy (TEM), Circular dichroism (CD), and Critical aggregation concentration (CAC). TEM detection: 10 μL of the compound solution (100 μM) was carefully dropped onto a copper grid. After 30 s, the excess solution was blotted with absorbent paper, and then the sample was negatively stained with uranyl acetate solution. Then, it was photographed with TEM. CD detection: The circular dichroism of three compound solutions (100 μM, 2 mL) was detected using a JASCO J-1500 CD spectrometer, and the recording range was from 185 nm to 280 nm. CAC detection: Three compound solutions with gradient concentrations (0.5 - 750 μM, 999 μL) were prepared, and pyrene acetone (0.1 mM, 1 μL) was added to each concentration solution. After mixing, it was placed in the dark at 37 °C for 6 hours. The fluorescence spectra of pyrene in each concentration solution were detected using a fluorescence spectrometer. Finally, the CAC value was calculated by comparing the fluorescence intensities of the third emission peak and the first emission peak (I3 / I1).

[0105] As Figure 4 shown in a, b, OTBS-FK-ER and OTBS-FR-ER self-assembled into nanofiber morphologies with a β-sheet structure with the assistance of the self-assembly unit "FFVLK", while there was no obvious assembly phenomenon for OTBS-R-ER. In addition, the Critical aggregation concentration (CAC) values of the three compounds were OTBS-FR-ER (63.6 μM), OTBS-FK-ER (76.9 μM), and OTBS-R-ER (308.7 μM), confirming that the "FFVLK" unit could effectively promote the concentration-dependent self-assembly of the chimeric peptide ( Figure 4 c).

[0106] Figure 5 To pass the total ROS indicator, O2 •- indicator, NO detection reagent, and ONOO -The indicator measured the ROS / RNS generation performance of three AIE probes (OTBS-R-ER, OTBS-FK-ER, and OTBS-FR-ER) in solution. Total ROS detection: The total ROS levels generated by the three AIE probes (OTBS-FR-ER, OTBS-FK-ER, and OTBS-R-ER) were evaluated using the fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). In an aqueous solution, the OTBS-R-ER solution (100 µM), OTBS-FK-ER solution (100 µM), and OTBS-FR-ER solution (100 µM) were mixed with DCFH-DA to make the working concentration of the three AIE probes reach 20 µM. Under white light irradiation (0.25 W cm -2 ), the fluorescence spectra were measured using a fluorescence spectrometer to evaluate the ability of each AIE probe to generate ROS at different irradiation times. O2 •- and ONOO - detection: Similar to the detection of total ROS, dihydrorhodamine 123 (DHR 123) was used to evaluate the generation of O2 •- , and the O56 probe was used to detect the generation of ONOO - . All samples were diluted from 100 µM to a working concentration of 20 µM. Under white light irradiation (0.25 W cm -2 ), the fluorescence spectra were measured using a fluorescence spectrometer to evaluate the ability of each AIE probe to generate O2 •- and ONOO - at different irradiation times. NO detection: A nitric oxide detection kit was used to detect the nitric oxide generation ability of the three AIE probes (OTBS-FR-ER, OTBS-FK-ER, and OTBS-R-ER). The standards (1 - 100 μM) were diluted with an aqueous solution at room temperature. After diluting the AIE probes from 100 µM to 20 µM concentration, 50 µL of the standards and AIE probes were added to a 96-well plate using a pipette. Finally, 50 µL of Gris Reagent I and 50 µL of Gris Reagent II were added to each well. Under white light irradiation (0.25 W cm -2 ), a multi-functional microplate reader was used to detect the absorbance at 540 nm of each AIE probe at different irradiation times to evaluate the NO generation ability.

[0107] Such as Figure 5As shown in a and b, the total ROS production of AIE probes OTBS-FR-ER and OTBS-FK-ER after 90 seconds of white light irradiation was 1.96 times and 2.54 times that of AIE probe OTBS-R-ER, respectively, and the O2 •- production after 5 minutes of white light irradiation was 1.85 times and 3.52 times that of AIE probe OTBS-R-ER, respectively. As Figure 5 shown in c and d, significant NO and ONOO - generation was also observed in the solutions of AIE probes OTBS-R-ER and OTBS-FR-ER containing the NO generation unit "RRRR". Compared with AIE probe OTBS-R-ER, the ONOO - generation in the self-assembled AIE probe OTBS-FR-ER nanofiber solution was significantly higher, which was attributed to the more excellent ROS generation ability brought by the self-assembled characteristics. In contrast, the ONOO - generated by OTBS-FK-ER lacking the NO donor unit "RRRR" was negligible.

[0108] Figure 6 To observe the ER targeting ability of three AIE probes (OTBS-R-ER, OTBS-FK-ER, and OTBS-FR-ER) in mouse prostate cancer RM-1 cells by CLSM. First, 8 × 10 4 RM-1 cells were seeded on a confocal culture dish. After the cells adhered, the culture medium was replaced with a culture medium containing the AIE probe (20 μM) and cultured at 37 °C for 6 hours. Then, the culture medium was changed to a serum-free medium containing the endoplasmic reticulum probe ER-tracker and cultured at 37 °C for 30 minutes. Next, the confocal culture dish was washed three times with PBS, and finally observed and photographed under CLSM (OTBS: excited at 633 nm; ER-tracker: excited at 488 nm). The results showed that the red fluorescence signal of the three AIE probes OTBS-FR-ER had a high degree of co-localization with the green fluorescence signal of ER-tracker, indicating that all three AIE probes could target the ER under the action of the ER targeting signal peptide "KDEL" ( Figure 6 a-c).

[0109] Figure 7 To observe the ROS / RNS production performance of three AIE probes (OTBS-R-ER, OTBS-FK-ER, and OTBS-FR-ER) in RM-1 cells by CLSM. First, 8 × 10 4RM-1 cells were seeded on confocal dishes. After the cells adhered, the culture medium was replaced with a medium containing the AIE probe (20 μM) and the cells were further cultured at 37 °C for 6 h. Then, the culture medium was replaced with a serum-free medium containing the total ROS probe DCFH-DA, O2 •- probe DAF-FM and ONOO - probe O56, and the cells were cultured at 37 °C for 30 min. Subsequently, the confocal dishes were washed three times with PBS and then placed under a CLSM (OTBS: excited at 633 nm; DCFH-DA / DAF-FM / O56: excited at 488 nm) for observation and photography after white light irradiation (0.25 W cm -2 , 3 min). Control: treated with PBS. L: white light irradiation, 0.25 W cm -2 , 3 min.

[0110] As Figure 7 shown in a, the total ROS generation levels of the self-assembled AIE probes OTBS-FR-ER and OTBS-FK-ER were relatively high under white light irradiation, which was attributed to the formation of nanofibers dominated by the self-assembly unit "FFVLK". After white light irradiation, the ROS fluorescence signal of the self-assembled AIE probe OTBS-FR-ER decreased slightly compared with that of OTBS-FK-ER, which might be due to the consumption of some ROS during the RNS generation process. As Figure 7 shown in b, obvious NO fluorescence signals were observed for the AIE probes OTBS-R-ER and OTBS-FR-ER under white light irradiation, and the NO fluorescence signal of the OTBS-FR-ER + L group was significantly higher. Similarly, under the synergistic action of the AIE probes OTBS, FFVLK, and RRRR, the ONOO - generation amount in the OTBS-FR-ER + L group was the highest ( Figure 7 c).

[0111] Figure 8 To observe the morphological changes of the endoplasmic reticulum of RM-1 cells induced by OTBS-FR-ER under light through a biological transmission electron microscope (Bio-TEM). First, RM-1 cells were seeded on 10 cm dishes. After the cells adhered, the old culture medium was discarded and replaced with a serum-free medium containing the AIE probe OTBS-FR-ER, and the cells were further cultured at 37 °C for 6 h. After 6 h, the serum-free RPMI-1640 medium was removed and replaced with a complete RPMI-1640 medium. The light irradiation group needed to be additionally irradiated with white light (0.25 W cm -2, treatment for 3 minutes). After 12 hours, discard the old medium and wash 3 times with PBS. Gently scrape the cells with a cell scraper and collect them into a 1.5 mL EP tube. Centrifuge to collect the cells at the bottom of the tube, discard the supernatant, and slowly add glutaraldehyde fixative along the tube wall. Place at 4 °C overnight. After dehydration, embedding, sectioning and staining of the samples, observe them under a TEM microscope. Control: PBS treatment. L: White light irradiation, 0.25 W cm -2 , 3 minutes. Red arrow: Swollen and vacuolated ER.

[0112] As Figure 8 shown, OTBS-FR-ER + L treatment induced significant swelling and vacuolization of the ER, confirming the induction of ER stress. In contrast, almost no changes were observed in the ER morphology of the Control group and the OTBS-FR-ER alone treatment group.

[0113] Figure 9 Evaluate the killing effects of three AIE probes (OTBS-R-ER, OTBS-FK-ER, and OTBS-FR-ER) on RM-1 tumor cells through a cytotoxicity experiment (CCK-8). Seed 2 × 10 3 RM-1 tumor cells in a 96-well plate. After the cells adhered, replace the medium with serum-free medium containing different concentrations of the three AIE probes and continue to culture at 37 °C for 6 hours. After 6 hours, remove the serum-free RPMI-1640 medium and replace it with complete RPMI-1640 medium. The light irradiation group needs to be additionally treated with white light irradiation (0.25 W cm -2 , 3 minutes). After 12 hours, detect the absorbance at 450 nm with a CCK-8 reagent to evaluate cell viability. L: White light irradiation, 0.25 W cm -2 , 3 minutes. The results showed that as the concentrations of the three AIE probes (OTBS-R-ER, OTBS-FK-ER, and OTBS-FR-ER) increased, the killing ability against RM-1 tumor cells increased. Among them, the cell killing effect of the OTBS-FR-ER + L group was always better than the other two groups, which may be due to the synergistic effect of ROS and RNS.

[0114] Figure 10 To explore the effect of three AIE probes (OTBS-R-ER, OTBS-FK-ER, and OTBS-FR-ER) on inducing apoptosis of RM-1 cells under light through a protein immunoblotting experiment (Western blot). Seed 2 × 10 5RM-1 tumor cells were seeded in 6-well plates. After the cells adhered to the wall, the culture medium was replaced with serum-free medium containing three AIE probes (20 µM) and further cultured at 37 °C for 6 hours. After 6 hours, the serum-free RPMI-1640 medium was removed and replaced with complete RPMI-1640 medium. The light irradiation group needed to be additionally treated with white light irradiation (0.25 W cm -2 , for 3 minutes). After 12 hours, the cells in each group were collected and proteins were extracted for Western blot verification. After loading the proteins, electrophoresis was carried out at a constant voltage of 150 V, and then transferred to the membrane at a constant current of 400 mA. The PVDF membrane was placed in the blocking solution and blocked for 1 hour. The PVDF membrane was washed three times with TBST. The primary antibody (1:1000) was incubated overnight at 4 °C. The next day, the primary antibody was recovered, and the PVDF membrane was washed three times with TBST. The secondary antibody (1:10000) was incubated at room temperature for 1 hour. The membrane was placed in a Western blot developer, and an enhanced chemiluminescence substrate developer was evenly dropped on the membrane for exposure. G1: Control, G2: OTBS-R-ER, G3: OTBS-FK-ER, G4: OTBS-FR-ER, G5: + L, G6:OTBS-R-ER + L, G7: OTBS-FK-ER + L, G8: OTBS-FR-ER + L. Control: Treated with PBS. L: White light irradiation, 0.25 W cm -2 , for 3 minutes.

[0115] As Figure 10 shown in a-c, after white light irradiation (0.25 W cm -2 , for 3 minutes), the protein expression levels of apoptosis-related markers (bax and cleaved-caspase-3) in the AIE probe treatment groups were significantly increased. Compared with the OTBS-R-ER + L group and the OTBS-FK-ER + L group, the expression levels of bax and cleaved-caspase-3 in the OTBS-FR-ER + L group were the highest, indicating that the synergistic effect of ROS and RNS generated by OTBS-FR-ER + L treatment induced a higher level of apoptosis

[0116] Figure 11 To explore the levels of ICD induced by three AIE probes (OTBS-R-ER, OTBS-FK-ER, and OTBS-FR-ER) in RM-1 cells under light by flow cytometry and ELISA experiments. To detect the release of Ecto-CRT, 1 × 10 5RM-1 tumor cells were seeded in 6-well plates. After the cells adhered to the wall, the culture medium was replaced with serum-free medium containing three AIE probes (20 µM) and continued to be cultured at 37 °C for 6 hours. After 6 hours, the non-irradiation group was not treated, and the irradiation group needed to be additionally treated with white light irradiation (0.25 W cm -2 , for 3 minutes). After 12 hours of irradiation, the old culture medium was discarded, washed 3 times with PBS, 1.5 mL of culture medium was added, and the cells were digested with trypsin. The cells were centrifuged at 1500 rpm at 4 °C for 5 min, the supernatant was removed, 200 μL of PBS solution containing calreticulin antibody (diluted 1:200) was added, and incubated at room temperature for 30 minutes. The cells were centrifuged at 1500 rpm at 4 °C for 5 min, the supernatant was removed, 200 μL of PBS solution containing AlexaFluor 647-labeled secondary antibody (diluted 1:400) was added, and incubated at room temperature for 50 minutes. After completion, PBS was added to terminate the staining, the supernatant was removed after centrifugation, 200 μL of PBS solution was added to resuspend, and the detection was carried out on a flow cytometer. For the detection of the release of extracellular high mobility group protein B1 (HMGB1), the cell seeding, drug addition and light irradiation processes were as above. After 12 hours of light irradiation, the supernatant of the culture medium was collected and centrifuged on a centrifuge (4 °C, 13500 g, 10 minutes). The release amount of HMGB1 in the supernatant was measured using an HMGB1 ELISA kit. Control: PBS treatment. L: White light irradiation, 0.25 W cm -2 , for 3 minutes.

[0117] As Figure 11 shown in a, all three AIE probes induced significant Ecto-CRT expression after light irradiation. Compared with the OTBS-R-ER + L group and the OTBS-FK-ER + L group, the OTBS-FR-ER + L group showed the most significant increase in Ecto-CRT level. To further evaluate ICD, we also analyzed the release of another marker of ICD, HMGB1. Consistent with the ecto-CRT results, after treatment with the three self-assembled AIE probes, the HMGB1 concentration in the collected cell supernatants increased significantly, and the HMGB1 concentration in the OTBS-FR-ER + L treatment group increased most significantly ( Figure 11 b). The above results confirmed that OTBS-FR-ER + L treatment could cause ER stress through the synergistic effect of ROS and RNS after treatment, inducing efficient ICD.

[0118] Figure 12To investigate the effect of three AIE probes (OTBS-R-ER, OTBS-FK-ER, and OTBS-FR-ER) on enhancing the immunogenicity of RM-1 cells under light through in vitro dendritic cell maturation experiments. First, bone marrow-derived dendritic cells (BMDCs) were isolated from the tibias and fibulas of 6-week-old male C57BL / 6 mice. After extraction, the BMDCs were filtered, centrifuged, and treated with red blood cell lysis. Then the BMDCs were cultured in RPMI-1640 complete medium containing GM-CSF (20 ng mL -1 ) and IL-4 (10 ng mL -1 ) for 5 days. On the 6th day, the induced and differentiated immature BMDCs were collected and re-plated in a 12-well plate. RM-1 cells were placed in the upper layer of the transwell chamber. After cell attachment, they were co-cultured with the three AIE probes (20 µM) (OTBS-R-ER, OTBS-FK-ER, and OTBS-FR-ER). After 6 hours, the medium was discarded and fresh medium was added. The non-irradiation group was not treated, and the irradiation group was additionally treated with white light irradiation (0.25 W cm -2 , 3 minutes). Subsequently, the above transwell chambers were moved above the 12-well plate containing BMDCs. After co-culturing for 24 hours, the BMDCs were collected for flow cytometry staining and analysis. Flow cytometry was used to evaluate the expression levels of mature BMDCs (CD11c + CD80 + CD86 + cells) in each treatment group. Control: Treated with PBS. L: White light irradiation, 0.25 W cm -2 , 3 minutes.

[0119] As shown in Figure 12 a, b, compared with the control group, RM-1 cells in the OTBS-R-ER + L, OTBS-FK-ER + L, and OTBS-FR-ER + L treatment groups could significantly promote the maturation of BMDCs (CD11c + CD80 + CD86 + cells), and the results were increased by 1.23-fold, 1.43-fold, and 1.61-fold, respectively. Consistent with the results of ICD level evaluation, RM-1 cells treated with the AIE probe OTBS-FR-ER + L induced the most obvious maturation of DCs, which represented that OTBS-FR-ER + L could induce more significant ICD through the synergistic induction of ER stress by ROS and RNS, thereby more effectively promoting the maturation of DCs.

[0120] Figure 13 To evaluate the retention time of three AIE probes (OTBS-R-ER, OTBS-FK-ER, and OTBS-FR-ER) in RM-1 tumors of mice using a small animal in vivo fluorescence imaging system. RM-1 cells (60 µL, 2 × 10 6 cells) were injected into the left back of 4-week-old male C57BL / 6 mice to establish a subcutaneous tumor mouse model of PCa. The tumor volume was continuously observed, and when the tumor volume reached approximately 80 mm 3 , the imaging experiment was carried out. The three AIE probes (100 µM, 100 µL) were respectively injected into the tumors, and the fluorescence intensities at the tumor sites of the mice were recorded at 0 h, 2 h, 4 h, 8 h, and 12 h after injection using a small animal in vivo fluorescence imaging system.

[0121] As Figure 13 shown in a, b, the fluorescence intensity of the AIE probe OTBS-R-ER group reached the peak 2 h after intratumoral injection, and then the fluorescence signal rapidly decayed, and the fluorescence signal intensity was negligible after 12 h. In contrast, the self-assembled AIE probes OTBS-FK-ER and OTBS-FR-ER showed continuous fluorescence enhancement, reached the peak at 8 h, and still showed strong fluorescence signals at 12 h, which was mainly due to the self-assembled unit "FFVLK" promoting the formation of the nanostructure of the nanoassemblies, thereby achieving long retention of the photosensitizer in the tumor.

Claims

1. An ER-targeted self-assembled AIE probe with cascaded ROS / RNS generation performance, characterized in that, The AIE probe includes four functional units: the AIE photosensitizer molecule OTBS, the amyloid-beta-derived amino acid sequence FFVLK, the oligomeric arginine unit RRRR, and the ER targeting signal peptide KDEL. Its molecular structure is as follows: 。 2. The preparation method of the ER-targeted self-assembled AIE probe with cascaded ROS / RNS generation performance according to claim 1, wherein, It includes the following steps: Dissolve the polypeptide N3-FFVLKRRRRKDEL and OTBS in dimethyl sulfoxide, and adjust the pH value of the solution to 8-9 with N,N-diisopropylethylamine; add copper(II) sulfate pentahydrate and sodium ascorbate to initiate the click chemical reaction, stir at room temperature in a nitrogen environment, and after the reaction is completed, purify using HPLC to obtain the product OTBS-FR-ER, that is, an ER-targeted self-assembled AIE probe with cascaded ROS / RNS generation performance.

3. The preparation method of the ER-targeting self-assembled AIE probe with cascaded ROS / RNS generation performance according to claim 2, characterized in that, The molar ratio of the polypeptide N3-FFVLKRRRRKDEL to OTBS is 1.5-2:

1.

4. The preparation method of the ER-targeted self-assembled AIE probe with cascaded ROS / RNS generation performance according to claim 2, characterized in that The preparation method of the OTBS includes the following steps: (1) Dissolve 4-bromo-N,N-bis(4-methoxyphenyl)aniline, bis(pinacolato)diboron, and tetrakis(triphenylphosphine)palladium in tetrahydrofuran; then add an aqueous solution of K2CO3; under a nitrogen environment, heat the mixture to 70-90 °C and reflux for 7-9 hours; after the reaction is completed, extract the mixture with a saturated sodium chloride aqueous solution, collect the organic phase; after rotary evaporation and purification, obtain 4-borate-4',4'-dimethoxytriphenylamine; (2) Dissolve 4-borate-4',4'-dimethoxytriphenylamine, 7-bromo-benzo[c][1,2,5]thiadiazole-4-carbaldehyde, and tetrakis(triphenylphosphine)palladium in tetrahydrofuran; then add an aqueous solution of K2CO3, and under a nitrogen environment, heat the mixture to 70-90 °C and reflux for 7-9 hours; after the reaction is completed, extract the mixture with a saturated NaCl aqueous solution, collect the organic phase, and after rotary evaporation and purification, obtain 7-(4-(bis(4-methoxyphenyl)amino)benzo)benzo[c][1,2,5]thiadiazole-4-carbaldehyde; (3) Dissolve 7-(4-(bis(4-methoxyphenyl)amino)benzo)benzo[c][1,2,5]thiadiazole-4-carbaldehyde, 2-(4-(pyridin-4-yl)phenyl)acetonitrile, and sodium ethoxide in absolute ethanol, stir at room temperature in a nitrogen environment, and after the reaction is completed, after rotary evaporation and purification, obtain 3-(7-(4-(bis(4-methoxyphenyl)amino)phenyl)benzo[c][1,2,5]thiadiazole-4-yl)-2-(4-(pyridin-4-yl)phenyl)acrylonitrile; (4) Dissolve 3-(7-(4-(bis(4-methoxyphenyl)amino)phenyl)benzo[c][1,2,5]thiadiazol-4-yl)-2-(4-(pyridin-4-yl)phenyl)acrylonitrile and 6-iodo-1-hexyne in N,N-dimethylformamide solution, heat to 70 - 90 °C under a nitrogen atmosphere and reflux for 12 - 20 hours; after the reaction is completed, perform rotary evaporation and purification. Dissolve the product in methanol, add an aqueous solution of potassium hexafluorophosphate, stir at room temperature, wash the resulting mixture with dichloromethane and perform extraction, retain the organic phase and perform rotary evaporation to obtain the hexafluorophosphate of OTBS.

5. The preparation method of the ER-targeted self-assembled AIE probe with cascaded ROS / RNS generation performance according to claim 4, characterized in that, The molar ratio of 4-bromo-N,N-bis(4-methoxyphenyl)aniline, bis(pinacolato)diboron and tetrakis(triphenylphosphine)palladium is 0.5 - 1: 1: 0.01; The molar ratio of 4-boronate-4',4'-dimethoxytriphenylamine, 7-bromo-benzo[c][1,2,5]thiadiazole-4-carbaldehyde and tetrakis(triphenylphosphine)palladium is 0.5 - 1: 1: 0.

01.

6. The preparation method of the ER-targeting self-assembled AIE probe with cascaded ROS / RNS generation performance according to claim 4, characterized in that, 7-(4-(bis(4-methoxyphenyl)amine)benzo) The molar ratio of benzo[C][1,2,5]thiadiazole-4-carbaldehyde, 2-(4-(pyridin-4-yl)phenyl)acetonitrile and sodium ethoxide is 1: 1 - 1.5: 0.

1.

7. The preparation method of the ER-targeted self-assembled AIE probe with cascaded ROS / RNS generation performance as described in claim 4, characterized in that, The molar ratio of 3-(7-(4-(bis(4-methoxyphenyl)amino)phenyl)benzo[c][1,2,5]thiadiazol-4-yl)-2-(4-(pyridin-4-yl)phenyl)acrylonitrile and 6-iodo-1-hexyne is 0.5 - 1:

1.

8. The preparation method of the ER-targeted self-assembled AIE probe with cascaded ROS / RNS generation performance according to claim 2, characterized in that, The preparation method of polypeptide N3-FFVLKRRRRKDEL comprises the following steps: (1) Dissolve dichlororesin in dichloromethane, place it in a solid-phase tube for swelling, and after completion, squeeze out dichloromethane with an ear bulb; (2) Weigh Fmoc-protected leucine and dissolve it in N,N-dimethylformamide, add N,N-diisopropylethylamine to adjust the pH to 8 - 9, add it into the solid-phase tube, and react on a shaker at room temperature; (3) Wash with dichloromethane, add a blocking solution for blocking, and after completion, wash successively with dichloromethane and N,N-dimethylformamide respectively; (4) Add an N,N-dimethylformamide solution containing piperidine to cleave the Fmoc protecting group, and after completion, wash with N,N-dimethylformamide; (5) Dissolve Fmoc-protected glutamic acid and O-benzotriazole-tetramethyluronium hexafluorophosphate in N,N-dimethylformamide, add N,N-diisopropylethylamine to adjust the pH to 8 - 9, add it into the solid-phase tube, and react on a shaker at room temperature; (6) Repeat steps (4) and (5), and successively add Fmoc-protected aspartic acid, Fmoc-protected lysine, Fmoc-protected arginine, Fmoc-protected arginine, Fmoc-protected arginine, Fmoc-protected arginine, Fmoc-protected lysine, Fmoc-protected leucine, Fmoc-protected valine, Fmoc-protected phenylalanine, Fmoc-protected phenylalanine and azidoacetic acid; (7) Add a polypeptide cleavage solution to cleave the polypeptide, collect the cleavage solution in a round-bottom flask, wash it with a dichloromethane solution containing trifluoroacetic acid, evaporate the collected solution to dryness using a rotary evaporator, add anhydrous ether to the dried substance, a solid will precipitate, blow dry the ether, and collect the solid; (8) Purify the product using high-performance liquid chromatography, and after freeze-drying, obtain the polypeptide N3-FFVLKRRRRKDEL.

9. The preparation method of the ER-targeted self-assembled AIE probe with cascaded ROS / RNS generation performance according to claim 8, wherein, The molar ratio of dichloro resin to leucine is 1: 1.5~2.

5.

10. Use of the ER-targeted self-assembled AIE probe with cascaded ROS / RNS generation performance described in claim 1 in the preparation of optical diagnostic or therapeutic reagents for prostate tumors.

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