WO2023098007A1 - Intelligent conversion double-stimuli-responsive probe chelated with metal ions, and preparation method therefor and use thereof - Google Patents

Abstract

An intelligent conversion double-stimuli-responsive probe chelated with metal ions, and a preparation method therefor and the use thereof. The probe can be self-assembled into large nanoparticles in a water buffer solution, and the large nanoparticles are then converted into small nanoparticles under the action of LAP and GSH, so as to enhance the accumulation thereof in a tumor and the deep tissue penetration, thereby improving near-infrared imaging of the tumor in vivo. In addition, it can be found for the first time that this LAP/GSH-driven disassembly and size reduction method can remarkably activate the photodynamic effect of a therapeutic drug, so as to achieve effective imaging-guided PDT on liver tumors and reduce the side effects on normal tissues. The chelating probe Ce6-Leu@Mn ²⁺ can improve the oxygen supply to overcome hypoxia and enhance the generation of ROS under X-ray radiation, thereby performing effective MRI imaging-guided radiotherapy on human liver HepG2 tumors in living mice. Therefore, a size-convertible nanosystem of the chelating probe may provide a powerful technique for improving the drug delivery efficiency, thereby enhancing tumor diagnosis and treatment.

Description

A kind of intelligent conversion dual stimulus response type probe of chelating metal ions and its preparation method and application technical field

The invention belongs to the technical field of reassembly mediated by tumor microenvironment, and relates to an intelligent conversion dual stimulation-response probe for chelating metal ions, a preparation method and application thereof.

Background technique

With the rapid development of nanobiotechnology and nanomedicine, drug delivery systems based on dynamic nanoassembly have attracted great research interest and are considered as a promising means to enhance local drug concentration for effective cancer diagnosis and treatment . This nanosystem is expected to improve the specificity, accumulation, and retention time of antitumor drugs. Stimulus-induced self-assembly approaches, which allow local assembly of molecules at disease sites of interest, have proven to be effective approaches to achieve signal amplification for imaging, enhanced therapeutic efficacy, and improved biosafety. For example, enzyme-induced self-assembling supramolecular hydrogels developed by Xu and colleagues have demonstrated improved accumulation and retention of small peptides for enhanced cancer imaging and therapy. In addition, Rao and Liang's group innovatively proposed the concept of an enzyme/GSH-mediated self-assembly method based on biorthogonal CBT-Cys. However, most of these contrast agents are still in the preclinical research stage, lacking the experimental evaluation of biological toxicity, pharmacokinetics and distribution in vivo, and there is still a certain distance from clinical application.

technical problem

In order to overcome the above-mentioned problems existing in existing contrast agents, the present invention rationally designs and synthesizes a novel intelligent dual-stimuli-responsive therapeutic probe, which maintains a large initial size to prolong blood circulation, and then overexpresses in tumors Leucine aminopeptidase (LAP) and reduced glutathione (GSH) are reduced in size at the tumor site for enhanced tumor imaging and therapy. Probes can initially self-assemble into large nanoparticles (~80 nm). Once the nanoparticles reach the tumor microenvironment (TME) through the enhanced permeability and retention (EPR) effect, the leucine motif and disulfide bond are spontaneously cleaved by LAP and GSH, respectively, to generate a ring via an intermolecular CBT-Cys condensation reaction. dimer, which can trigger the in situ conversion of initially large nanoparticles to tiny nanoparticles (~23 nm). This LAP/GSH-driven in situ shape/size conversion approach has the following advantages: (1) achieves shape conversion to facilitate probe penetration into tumor tissue; (2) amplifies fluorescence and magnetic resonance signals and improves ¹ O ₂ generation, Guide PDT with enhanced NIR/MRI imaging; (3) Increase the O ₂ production of Ce6-Leu@Mn ²⁺ to improve the curative effect of radiotherapy (RT). Thus, the present LAP/GSH-responsive nanosystem overcomes the size dilemma faced by conventional nanomedicine and provides a powerful and surprising tool for precise cancer diagnosis and therapy.

technical solution

The present invention adopts the following technical scheme: an intelligent conversion dual-stimuli-response probe for chelating metal ions has the following chemical structural formula:

.

The application of the above-mentioned intelligent conversion dual stimulus-responsive probe for chelating metal ions in the preparation of reagents for tumor diagnosis and/or treatment.

The preparation method of the above-mentioned intelligent conversion dual stimuli-responsive probe for chelating metal ions comprises the following steps: (1) compound 1 is subjected to amide condensation reaction with NH ₂ -CBT to obtain compound 2; (2) compound 2 is removed from the protecting group to obtain Compound 3; (3) Compound 3 undergoes amide condensation reaction with N-fluorenylmethoxycarbonyl-S-tert-butylthio-L-cysteine to obtain compound 4; (4) Compound 4 removes the protecting group to obtain compound 5; (5) Compound 5 was reacted with a photosensitizer to obtain compound 6; (6) Compound 6 was deprotected to obtain compound 7; (7) Compound 7 was amide condensed with N-tert-butoxycarbonyl-L-leucine Compound 8 is obtained by reaction; (8) Compound 8 removes the protective group to obtain Ce6-Leu; (9) Mix Ce6-Leu and inorganic manganese salt in a solvent, add organic additives, and stir to obtain double stimulation of intelligent conversion of chelated metal ions Responsive probes.

Ce6-Leu has the following chemical structural formula:

.

In the above technical scheme, in step (1), the molar ratio of compound 1 to NH ₂ -CBT is 1:1.2; the amide condensation reaction is carried out in the presence of N-methylmorphine and isobutyl chloroformate; the amide condensation reaction is at room temperature React for 15 to 24 hours.

In the above technical scheme, in step (2), the deprotection group of compound 2 is carried out in N,N-dimethylformamide/piperidine mixed solvent; the volume of N,N-dimethylformamide and piperidine The ratio is 4:1.

In the above technical scheme, in step (3), the molar ratio of compound 3 to N-fluorenylmethoxycarbonyl-S-tert-butylthio-L-cysteine is 1:1.2; the amide condensation reaction is carried out in 1-hydroxybenzene Carried out in the presence of triazole, O-benzotriazole-tetramethyluronium hexafluorophosphate and diisopropylethylamine; the amide condensation reaction is carried out at room temperature for 2 to 4 hours.

In the above technical scheme, in step (4), the deprotection of compound 4 is carried out in a mixed solvent of dichloromethane/trifluoroacetic acid; the volume ratio of dichloromethane and trifluoroacetic acid is 4:1.

In the above technical solution, in step (5), the molar ratio of compound 5 to the photosensitizer is 1.1:1; the photosensitizer is NHS-activated chlorin E6 (Ce6-NHS).

In the above technical scheme, in step (6), the deprotection group of compound 6 is carried out in N,N-dimethylformamide/piperidine mixed solvent; the volume of N,N-dimethylformamide and piperidine The ratio is 4:1.

In the above technical scheme, in step (7), the molar ratio of compound 7 to N-tert-butoxycarbonyl-L-leucine is 1:1.2; Carry out in the presence of 3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and diisopropylethylamine; amide condensation reaction takes 8 to 12 hours at room temperature.

In the above technical solution, in step (8), the deprotection of compound 8 is carried out in a mixed solvent of dichloromethane/trifluoroacetic acid; the volume ratio of dichloromethane and trifluoroacetic acid is 4:1.

In the above technical solution, in step (9), the inorganic manganese salt is manganese chloride, the solvent is methanol, and the organic additive is pyridine; preferably, the stirring is at 35-40° C. for 3-5 hours. Preferably, the molar weight of the inorganic manganese salt is 4-6 times that of Ce6-Leu.

The chemical structural formula of NHS-activated photosensitizer chlorin E6 is as follows:

.

In the present invention, after the stirring in step (9), it is separated and purified by high-performance liquid chromatography (HPLC) to obtain an intelligent conversion dual-stimuli-responsive probe for chelating metal ions, which is a conventional technique. Preferably, the HPLC separation method is: C18 column, 3.5 μm, 4.6×100 mm; mobile phase: A is water; B is acetonitrile; flow rate: 3 mL/min; linear gradient elution program: 0 min, A:B = 95:5; 13 min, A:B = 0:100.

The probe of the present invention reassembles the nanoparticle probes into nanofibers through the dual stimulation of leucine aminopeptidase and glutathione overexpressed in the tumor microenvironment, and realizes the restoration of the fluorescence of the probes and the ability to generate ROS , so as to achieve tumor-specific fluorescence imaging and photodynamic therapy. Compared with optical imaging, magnetic resonance imaging (MRI) is a non-invasive imaging method with high spatial resolution and good penetration depth, and is an attractive diagnostic technology for clinical diagnosis and tumor monitoring. The Ce6 -Leu chelates manganese(II) ions (Mn ²⁺ ), which can be used as an MRI imaging agent.

Beneficial effect

Due to the application of the above-mentioned technical scheme, the present invention has the following advantages compared with the prior art: 2-cyanobenzothiazole and 1,2-aminothiol are used in the present invention to undergo rapid and efficient click condensation reactions to form amphiphilic Dimers, and through the change of intermolecular forces, the nanoparticles reassemble into nanofibers. When the probe enters the tumor cell, under the stimulation of the overexpressed leucine aminopeptidase and glutathione in the tumor cell, the original amino group and sulfhydryl group in the cysteine structure are exposed, thereby interacting with 2-cyanobenzene The cyano group of thiazole (CBT) undergoes a click condensation reaction, which is not affected by the external environment; the Mn ²⁺ chelating probe (Ce6-Leu@Mn ²⁺ ) is proved to be able to catalyze endogenous H ₂ O ₂ in anoxic The ability of the tumor site to continuously generate _O2 , thereby improving oxygen supply to enhance the effect of radiation therapy.

The diagnostic and therapeutic functions of the smart probes responding to the tumor microenvironment of the present invention can only be activated when triggered by a special tumor microenvironment. Interfering with diagnosis and treatment. Therefore, intelligent diagnosis and treatment reagents that respond to the tumor microenvironment can effectively improve the accuracy of cancer diagnosis and the effect of treatment.

Description of drawings

Figure 1 shows the MALDI-TOF/MS of Ce6-Leu@Mn ²⁺ and Ce6-Ac@Mn ²⁺ .

Fig. 2 is the ultraviolet-visible absorption spectrum of Ce6-Ac and Mn ²⁺ chelated Ce6-Ac.

Figure 3 shows the synthesis and MRI characterization of the Ce6-Leu@Mn ²⁺ probe, (a) the UV-Vis absorption spectrum of Ce6-Leu and Mn ²⁺ chelated Ce6-Leu (Ce6-Leu@Mn ²⁺ ), ( b) TEM image and (c) particle size distribution of Ce6-Leu@Mn2 ⁺ and Ce6-Leu@ ^Mn2+ treated with LAP and GSH, (d) T1-weighted MR image of Ce6-Leu@ ^Mn2+ with longitudinal relaxation (r1), (e) T1 MR signal changes of Ce6-Leu@Mn ²⁺ and Ce6-Ac@Mn ²⁺ with or without LAP and GSH treatment, (f) Mn ²⁺ labeled Ce6-Leu or Time dependence of Ce6-Ac (500 μM, 200 μL) T1-weighted MR images with tumor aggregation and (g) Quantitative MR signal intensity changes in the tumor (I/I ₀ ), where I is the MR signal at a specific time point and I ₀ is the anterior MR signals of mice at time points. Pre means ***P<0.001, **P<0.01, *P<0.05 in mice before Mn ²⁺ labeled probe treatment.

Figure 4 is the characterization of the catalase-like probe Ce6-Leu@Mn ²⁺ , (a) schematic diagram of Ce6-Leu@Mn ²⁺ nanoparticles, LAP/GSH triggers the reassembly of nanoparticles with smaller size and better catalase-like properties to alleviate tumor hypoxia and enhance radiotherapy efficacy. (b) Generation of O ₂ from H ₂ O ₂ (1 mM) in different solutions. (Inset: _H2O2 treatment with LAP and _GSH in Ce6-Leu@Mn2 ⁺ solution). (c) _O concentration of 40 μM Ce6-Leu@Mn2 ⁺ treated with LAP and _GSH with different concentrations of _H2O2 . (d) _O2 concentrations _of LAP and GSH treated with different concentrations of Ce6-Leu@Mn2 ⁺ under H2O2 (1 mM).

Figure 5 shows the toxicity of Ce6-Leu@Mn ²⁺ and Ce6-Ac@Mn ²⁺ to 3T3 cells.

Figure 6 shows that the Ce6-Leu@Mn ²⁺ probe enhances the efficiency of radiotherapy in vitro, (a) Confocal images of HepG2 cells treated with Hoechst 33342 (blue, nucleus) and hypoxia probe (red, hypoxic cells) Fluorescence image. Scale bar: 20 μm, (b) Ce6-Leu, Ce6-Ac@Mn ²⁺ or Ce6-Leu@Mn ²⁺ (30 μM) incubated for 4 hours, HIF-1α protein expression in HepG2 cells, (c) by γ-H2AX To evaluate the DNA damage of HepG2 cells by different treatments. Scale bar: 20 μm (d) ***P<0.001 for corresponding quantification of cell numbers per cell. (e) Cell migration assay of HepG2 cells treated with different probes under X-ray irradiation (0 and 6 Gy). (f) The radiosensitization effect of differently treated HepG2 cells was assessed by live/dead staining. Scale bar: 200 μm.

Figure 7 is the detection of cellular hypoxia.

Figure 8 is a quantitative analysis of cell migration.

Figure 9 is an in vivo enhanced radiation therapy study. (a) Time dependence of oxygenated hemoglobin (HbO ₂ , 850 nm) concentration in HepG2 tumors after intravenous injection of Ce6-Leu, Ce6-Ac@Mn ²⁺ or Ce6-Leu@Mn ²⁺ (200 μM, 200 μL) Changes and (b) corresponding quantitative analysis of _HbO PA intensity at the tumor site. (c) Immunofluorescence images of tumor sections from different groups, stained with anti-HIF-1α antibody. Nuclei were stained with DAPI (blue). Scale bar: 60 μm, (d) Tumor growth curves of mice receiving different treatments. (e) Photographs of dissected tumors in different groups at 14 days. (f) Tunnel staining and H&E staining of tumor tissues of different groups of mice sacrificed 48 hours after radiotherapy. Scale bar: 60 μm ***P<0.001, **P<0.01, *P<0.05.

Figure 10 shows in vivo PA images and PA signals of tumors at different time points after intravenous injection of Ce6-Leu, Ce6-Ac@Mn ²⁺ or Ce6-Leu@Mn ²⁺ .

Figure 11 shows immunofluorescence staining for tumor hypoxia assessment.

Fig. 12 is the photographs of mice on days 0, 2, 6, 10, and 14 after different treatments.

Figure 13 shows the average body weight and mean body weight of mice in each group.

Figure 14 is a schematic diagram of the preparation of Ce6-Leu and Ce6-Ac.

Embodiments of the present invention

The present invention has developed a leucine aminopeptidase and glutathione dual-response intelligent molecular probe integrating nuclear magnetic imaging and photodynamic therapy, which has great research and application value. Under the stimulation of the environment, the reassembly from spherical nanoparticles to nanofibers, and the ability to fluoresce and generate ROS are restored, thereby enabling specific fluorescence imaging and photodynamic therapy of tumors in vivo. This size-switchable nanosystem will provide a new advanced technology. , to improve drug delivery efficiency for precise tumor diagnosis and treatment.

Specifically, the steps of the method provided by the present invention are as follows: (1) Construction and synthesis of dual stimulus-responsive probes: according to the designed synthesis steps: first, compound 1 undergoes amide condensation reaction with NH ₂ -CBT, and then uses 20% The piperidine (N,N-dimethylformamide: piperidine = 4:1, v/v) removes the protecting group Fmoc; followed by N-fluorenylmethoxycarbonyl-S-tert-butylthio-L- Cysteine undergoes amide condensation reaction, followed by 20% trifluoroacetic acid (dichloromethane: trifluoroacetic acid = 4:1, v/v) to remove the Boc protecting group of the intermediate compound; The NHS-activated photosensitizer chlorin E6 was reacted, and the obtained intermediate compound was deprotected with 20% piperidine (N,N-dimethylformamide: piperidine = 4:1, v/v) The intermediate obtained by the group Fmoc reacts with N-tert-butoxycarbonyl-L-leucine to obtain the product, and then 20% trifluoroacetic acid (dichloromethane: trifluoroacetic acid = 4: 1, v/v) protects Boc The group was removed to obtain the final probe Ce6-Leu; (2) The dual stimuli-responsive probe Ce6-Leu@Mn ²⁺ for intelligent conversion of chelated metal ions: Dissolve Ce6-Leu and MnCl ₂ in methanol, and then Pyridine was added, stirred, and finally purified by HPLC to obtain the compound Ce6-Leu@Mn ²⁺ .

In vivo magnetic resonance imaging. HepG2 tumor-bearing mice were intravenously injected with Ce6-Leu@Mn ²⁺ or Ce6-Ac@Mn ²⁺ (500 μM), respectively added to 200 μL of PBS. Mice were then anesthetized with 3% isoflurane mixed with oxygen (0.5 L/min) and imaged using a 3.0 T clinical MR scanner (MR solutions, UK) equipped with a small animal imaging coil.

Detection of cellular hypoxia. HepG2 cells were seeded on glass bottom culture dishes at a density of 8 × ¹⁰³ cells per well overnight. Then, after 4 h of incubation with different reagents (30 μM), the hypoxia probe (hypoxia red detection reagent) was added to HepG2 cells and incubated at 37 °C for 30 min under ₅ % CO. Wash three times with PBS to remove excess reagent. Nuclei were then stained with Hoechst 33342 for 15 min and characterized by confocal laser scanning microscopy. (CLSM; λex = 596 nm, λem = 670 nm).

Determination of intracellular HIF-1α levels. Cellular HIF-1α levels were determined by western blot analysis. After 4 h incubation with different reagents (30 μM), HepG2 cells were washed 3 times with ice-cold PBS and lysed using RIPA lysis buffer containing complete protease inhibitors. 60 μg of protein in each sample was separated by SDS-PAGE and transferred to PVDF membrane. After blocking with 5% skim milk for 2 hr, PVDF membranes were incubated with HIF-1α antibody overnight at 4°C, followed by incubation with the corresponding secondary antibody-conjugated horseradish peroxidase for 2 hr at room temperature. PVDF membranes were observed with the ECL plus detection system.

DNA double-strand breaks and cell migration assays. For DNA double-strand break analysis, HpeG2 cells were seeded into 35 mm dishes at a density of 5 × ¹⁰⁴ and cultured overnight. The cells were divided into four groups: RT, Ce6-Leu+RT, Ce6-Ac@Mn ²⁺ +RT , Ce6-Leu@Mn ²⁺ +RT, and the medium containing different reagents (at a concentration of 30 μM) was added to the HpeG2 cells middle. After 4 hours of incubation, excess reagent was removed by washing three times with PBS. HpeG2 cells were then treated with X-rays at a dose of 6Gy. After treatment, cells were fixed with 4% paraformaldehyde for 0.5 hr, permeabilized with 1% Triton X-100 for 10 min to rupture the cell membrane, and then blocked with 5% BSA for 1 hr at 37°C. Afterwards, fixed cells were incubated with 400 μL of γ-H2AX antibody overnight at 4°C, and then incubated with secondary antibody Cy3-labeled goat anti-Rabbit IgG (H+L) for 1 h at 37°C after washing. Finally, nuclei were stained with Hoechst 33342 and then examined using an Olympus confocal microscope (Olympus, Tokyo, Japan) to analyze the red phosphorylated H2AX signal.

For cell migration assays, HpeG2 cells were seeded in 6-well plates overnight at 4 × ¹⁰⁵ cells per well. After reaching approximately 95% confluency, medium containing different reagents (at a concentration of 30 μM) was added to the HpeG2 cells. After 4 hours of incubation, excess reagent was removed by washing three times with PBS. HpeG2 cells were then treated with X-rays at a dose of 0 or 6 Gy, and the cell layer was scraped using a 1 mL sterile pipette tip to create a gap. Cell migration was quantified manually by photographing cells before and 120 hours after incubation.

In vivo PA imaging of tumors. In vivo PA imaging was performed using a real-time multispectral photoacoustic tomography system (MOST, Isera Medical, Germany). Mice bearing HepG2 subcutaneous tumors were injected intravenously with Ce6-Leu, Ce6-Leu@Mn ²⁺ or Ce6-Ac@Mn ²⁺ (200 μM, 200 μL). Mice were then anesthetized with isoflurane and placed in a water bath to maintain their body temperature at 34 °C for PA imaging by MSOT. After image reconstruction, the PA signal of _HbO2 at the tumor site was separated from the PA image with MSOT software.

Immunofluorescent staining was used to assess tumor hypoxia. Mice bearing HpeG2 tumors were randomly divided into 4 groups. After intravenous injection of PBS (200 μL), Ce6-Leu (200 μL, 200 μM), Ce6-Ac@Mn ²⁺ (200 μL, 200 μM) or Ce6-Leu@Mn ²⁺ (200 μL, 200 μM), tumors were dissected from mice and sectioned for Immunofluorescence staining for HIF-1α was performed. Sections were observed with a confocal laser microscope.

HE and TUNEL staining of tumors were accepted. HepG2 tumor-bearing mice (n=3) were intravenously injected with PBS (200 μL), Ce6-Leu (200 μL, 200 μM), Ce6-Ac@Mn ²⁺ (200 μL, 200 μM) or Ce6-Leu@Mn ²⁺ (200 μL, 200 μM ). The tumor was irradiated with 8Gy of X-rays 6h after injection. Forty-eight hours after the different treatments, tumors were dissected from mice and fixed in neutral buffered formalin (10%). Then, tumors were sectioned into 4 μm thick sections for hematoxylin-eosin (H&E) and TUNEL staining. Sections were then observed with a confocal laser microscope.

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these embodiments are only used to explain and describe the technical solutions in the present invention, and are not intended to limit the scope of the present invention. In addition, unless otherwise specified, the materials, reagents, instruments, etc. used in the following examples can be obtained through commercial means.

Example 1: Synthesis and characterization of the smart conversion dual stimuli-responsive probe Ce6-Leu@Mn ²⁺ for chelating metal ions and the control probe Ce6-Ac@Mn ²⁺ : (1) Compound 1 (400 mg , 0.85 mmol) was dissolved in 10 mL tetrahydrofuran, and N-methylmorphine (130 mg, 1.28 mmol) was added dropwise, then the round bottom flask was placed in an ice-salt bath, cooled to 0 ^o C, and then chlorine was added dropwise Isobutyl formate (175 mg, 1.28 mmol), after activation for half an hour, added 2-amino-6-cyanobenzothiazole (NH ₂ -CBT, 179 mg, 1.00 mmol) dissolved in dry tetrahydrofuran, kept React at 0 ^o C for 1 hour, then stir overnight at room temperature. After the reaction, the solvent was spin-dried by a rotary evaporator, and then the residual solid was redissolved in ethyl acetate (50 mL), and extracted three times with an aqueous solution of sodium bicarbonate. . Using petroleum ether (PE) and ethyl acetate (EA) = 2:1 volume ratio as the eluent, the crude product was purified by silica gel chromatography to obtain Intermediate 1; (2) Intermediate 1 (500 mg, 0.80 mmol) was dissolved in 8 mL DMF, and then the reaction flask was placed in an ice-water bath, and then 2 mL of piperidine was added dropwise and kept at 0 ^o C for 5 minutes. After the reaction, the solvent and piperidine were removed by rotary evaporation. Using dichloromethane (DCM) and methanol (MeOH) = 80:1 volume ratio as the eluent, the crude product was purified by silica gel chromatography to obtain intermediate 2; (3) Add the intermediate to a 20 mL round bottom flask Body 2 (250 mg, 0.62 mmol), then dissolved with dry DMF, then added HBTU (282.15 mg, 0.74 mmol), HOBT (100.44 mg, 0.74 mmol) and DIPEA (213.68 μL), stirred for 15 minutes and then The compound N-fluorenylmethoxycarbonyl-S-tert-butylthio-L-cysteine (321.04 mg, 0.74 mmol) was added. Continue to stir and react at room temperature for 2 hours. After the reaction, remove the solvent by rotary evaporation, then add 25 mL of ethyl acetate to redissolve the crude product, and then use 25 mL of ultrapure water, saturated sodium bicarbonate, and sodium chloride aqueous solution for the organic phase Wash each once. The organic phase was dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation, and the crude product was purified by silica gel chromatography with petroleum ether (PE) and ethyl acetate (EA) = 2:1 volume ratio as the eluent to obtain the intermediate 3; (4) Add intermediate 3 to 20 mL of DMF solution containing 20% (volume ratio) trifluoroacetic acid, react at room temperature for 1 hour, remove solvent and trifluoroacetic acid by rotary evaporation, and obtain intermediate 4 (its structure shown as compound 5 in Figure 1). Intermediate 4 was not further purified. Accurately weigh 40 mg of intermediate 4 (0.0558 mmol), add 20 mL of anhydrous DMF solution to dissolve, then add 45.71 mg of Ce6-NHS (0.05 mmol) and 7.76 mg of DIPEA (0.06 mmol), and stir at room temperature for 2 hours, Separation and purification were carried out by HPLC, and the components with absorption spectra at 400 nm were collected to obtain Intermediate 5; (5) Intermediate 5 (45 mg, 0.035 mmol) was dissolved in 8 mL DMF, and then the reaction bottle was placed in an ice-water bath 2 mL of piperidine was then added dropwise, and the reaction was maintained at 0 ^o C for 5 minutes. After the reaction, HPLC was used for separation and purification, and the components with absorption spectra at 400 nm were collected to obtain intermediate 6; (6) in 10 mL Intermediate 6 (26 mg, 0.025 mmol) was added to a round bottom flask, dissolved with 5 mL of anhydrous DMF, and then NHS-activated N-tert-butoxycarbonyl-L-leucine (9.85 mg, 0.03 mmol) was added , reacted at room temperature for 2 hours, then removed the solvent by rotary evaporation, and separated and purified by semi-preparative high-performance liquid chromatography to obtain intermediate 7; (7) Add intermediate 7 (19 mg, 0.015 mmol), after reacting at room temperature for 1 hour, the solvent and trifluoroacetic acid were removed by rotary evaporation, and Ce6-Leu was separated and purified by semi-preparative high-performance liquid chromatography; (8) Add the intermediate to a 10 mL round bottom flask 6 (26 mg, 0.025 mmol) was dissolved in 5 mL of anhydrous DMF, then acetic anhydride (3.06 mg, 0.03 mmol) was added, stirred at room temperature for 2 hours, then the solvent was removed by rotary evaporation, and Ce6-Ac was purified and isolated by HPLC ; The above steps (1) to (8) are consistent with the submitted application CN2021113897458 (a dual stimulus-responsive probe of leucine aminopeptidase and glutathione and its preparation method and application), chemical structure and characterization Reference can be made to this application, and the schematic reaction process of the present invention is described in FIG. 14 .

(9) Dissolve 0.0084 mmol Ce6-Leu and MnCl ₂ (5.28 mg, 0.042 mmol) in 1 mL methanol, then add 100 μL pyridine, stir at 37 °C for 4 hours, and finally purify by HPLC to obtain the compound Ce6-Leu@ Mn ²⁺ (8.85 mg, 85%). MS (MALDI-TOF) Calcd for: C ₆₁ H ₇₃ MnN ₁₁ O ₈ S ₃ ([M+Na] ⁺ ): 1262.440, found: 1262.793; Ce6-Leu was replaced by Ce6-Ac to obtain Ce6-Ac@Mn ²⁺ (7.84 mg 80%). MS (MALDI-TOF) Calcd for: C ₅₇ H ₆₄ MnN ₁₀ O ₈ S ₃ ([M] ⁺ ): 1167.350, found: 1167.833.

Fig. 1 shows the structural formula and mass spectrum of the above-mentioned Ce6-Leu@Mn ²⁺ and Ce6-Ac@Mn ²⁺ .

Example 2 Ce6-Leu@Mn ²⁺ performance research: Figure 2 is the absorption spectrum of Ce6-Ac before and after chelating manganese ions, and Figure 3 is the related test of the probe and the comparison probe. The results show that Ce6-Leu and Ce6- Ac has a high-intensity initial peak at 410nm, and after chelating with Mn ²⁺ , the intensity of this peak is significantly suppressed, and a slight blue shift appears at the same time (Fig. 2, Fig. 3a), which indicates that Mn ²⁺ has been Successfully incorporated into Ce6-Leu or Ce6-Ac to obtain Ce6-Leu@Mn ²⁺ , Ce6-Ac@Mn ²⁺ . The morphology of Ce6-Leu@Mn ²⁺ was studied by transmission electron microscopy (TEM). It can self-assemble into large particles with an average particle size of 59.44±7.83nm in PBS buffer. After treatment with LAP and GSH, it can be decomposed into Small nanoparticles with a size of 24.13 ± 2.73 nm (Fig. 3b and 3c). The T1 magnetic resonance signal of Ce6-Leu@Mn ²⁺ gradually increased in a concentration-dependent manner, and its T1 relaxivity (r1) was measured and calculated to be 4.23 mM ^-1 S ^-1 (Fig. 3d), Ce6-Ac@Mn ² The T1 magnetic resonance signal of ⁺ also increased gradually in a concentration-dependent manner, and its T1 relaxivity (r1) was measured and calculated to be 3.50 mM ^-1 S ^-1 . Moreover, once the Ce6-Leu@Mn ²⁺ solution was treated with LAP and GSH, the T1 MR signal was 1.62 times higher than that of the solution without LAP and GSH treatment (Fig. 3e). However, almost no T1 MR signal changes were observed for the control probe Ce6-Ac@Mn ²⁺ treated with or without LAP and GSH; therefore, Ce6-Leu@Mn ²⁺ is a promising T1-weighted contrast agent, In vivo MRI imaging of tumors.

The in vivo MRI performance of the Ce6-Leu@Mn ²⁺ probe was further evaluated. Figure 3f shows mice with subcutaneous HepG2 tumor xenografts (n=3) at selected time points after intravenous injection of probes Ce6-Leu@Mn ²⁺ , Ce6-Ac@Mn ²⁺ (200 µL, 500 µM) A series of representative MR images obtained, the T1 MR signal intensity of the mouse tumor after injection of Ce6-Leu@Mn ²⁺ gradually increased over time, and reached a plateau at 4 hours after injection, which was significantly higher than that of Ce6-Ac@ Mn ²⁺ (Fig. 5g). In addition, high-resolution images showed that the enhanced MR signal of Ce6-Leu@Mn ²⁺ mice was almost distributed throughout the tumor tissue, indicating that LAP/GSH-driven size reduction of Ce6-Leu@Mn ²⁺ can significantly improve tumor penetration ability. Collectively, these evidences strongly demonstrate the great potential of Ce6-Leu@Mn ²⁺ for precise visualization of tumors in vivo.

The catalytic ability of Mn ²⁺ chelated Ce6 derivatives to convert H ₂ O ₂ to O ₂ is closely related to the size of nanoparticles, and severe self-aggregation will weaken the catalytic effect of nanoparticles. Next, the evaluation probe Ce6-Leu@Mn ²⁺ exhibited catalase-like activity to convert H ₂ O ₂ to O ₂ , that is, Ce6-Leu@Mn ²⁺ solution (40 µM) in the presence or absence Treated with _H2O2 (1 mM) in the presence of LAP and GSH, then _O2 generation was measured using a dissolved oxygen meter (Fig _. 4a). As shown in Figure 4b, Ce6-Leu@Mn ²⁺ treated with LAP and GSH (expressed as Ce6-Leu@Mn ²⁺ +LAP+GSH+H ₂ O ₂ ) compared with the control Ce6-Ac@Mn ²⁺ , Significant O ₂ production was shown, indicating that the reaction of Ce6-Leu@Mn ²⁺ to LAP and GSH can generate a large amount of O ₂ , and the amount of production is positively correlated with the amount of H ₂ O ₂ used (Fig. 4c). Similarly, under the condition that the amount of H ₂ O ₂ (1 mM) was constant, the O ₂ generation was gradually enhanced with the increase of the probe Ce6-Leu@Mn ²⁺ concentration (Fig. 4d). Taken together, these results highly demonstrate that the probe Ce6-Leu@Mn ²⁺ enhances the ability to generate O ₂ under the action of LAP and GSH and is used to overcome tumor hypoxia to enhance cancer radiotherapy.

The ability of Ce6-Leu@Mn ²⁺ for converting H ₂ O ₂ to O ₂ in HepG2 cells was further evaluated. First, the MTT method was used to detect the proliferation of 3T3 cells by Ce6-Leu@Mn ²⁺ and Ce6-Ac@Mn ²⁺ . Toxicity is negligible. Hypoxia/oxidative stress detection kit was used to detect intracellular hypoxia, and the fluorescent signal of hypoxia probe (hypoxia red detection reagent) was detected by CLSM method in cells with different treatments. As shown in Figure 6a, strong red fluorescence was detected in the two groups of cells treated with PBS and Ce6-Leu respectively, indicating that the intracellular environment was highly hypoxic. In contrast, HepG2 cells receiving Ce6-Ac@Mn ²⁺ showed relatively low fluorescence, while cells containing Ce6-Leu@Mn ²⁺ showed the weakest fluorescence even for 8 hours (Fig. 7), indicating that O ₂ The production of significantly alleviated cellular hypoxia. In addition, the intracellular expression level of HIF-1α was also determined by western blot (WB) analysis, which can be degraded by some enzymes under normoxic conditions, but remains highly expressed under hypoxic conditions. Figure 6b clearly shows that the expression of HIF-1α in HepG2 cells is significantly inhibited after treatment with Ce6-Leu@Mn ²⁺ , which proves again that Ce6-Leu@Mn ²⁺ can generate oxygen in cancer cells and effectively relieve intracellular lack of oxygen. Cells were analyzed for DNA double-strand breaks by γ-H2AX immunofluorescent staining. The results presented in Figures 6c and 6d show that under Ce6-Leu@Mn ²⁺ and X-ray (6 Gy) treatment (indicated as Ce6-Leu@Mn ²⁺ +RT), distinct DNA was detected in HepG2 cells injury and X-ray irradiation, while only a slight increase in γ-H2AX was observed in Ce6-Ac@Mn ²⁺ and X-ray irradiated (6 Gy) (expressed as Ce6-Ac@Mn ²⁺ +RT) cells, and in X-ray Almost no γ-H2AX was detected in cells treated with X-ray radiation (indicated as RT) and PBS or Ce6-Leu (indicated as Ce6-Leu+RT). Next, the metastatic and cell migration abilities of the differently treated cells were investigated. As shown in Figure 6e, all four groups of HepG2 cells showed good proliferation and migration capabilities within 120 hours without X-ray irradiation; however, after X-ray (6 Gy) irradiation, cells containing Ce6-Leu@Mn The ²⁺ cells still retain about 20% of the cell migration ability, which is significantly lower than other control groups (Figure 8). Furthermore, the radiosensitizing effect of X-ray radiation in the presence of Ce6-Leu@Mn2 ⁺ was evaluated in live cells by live/dead assay. As shown in Figure 6f, a large number of dead cells (red fluorescence) were clearly detected in the Ce6-Leu@Mn ²⁺ +RT cells, while only a small number of dead cells were observed in the cells of the other four control groups. Collectively, these results strongly demonstrate that the probe Ce6-Leu@Mn2 ⁺ exhibits good cytotoxicity against cancer cells under X-ray irradiation.

It is well known that solid tumors are generally insensitive to radiotherapy. The radiosensitizing effect of the probe of the present invention improves the radiotherapy effect of BALB/c mice bearing HepG2 tumors. The performance of the Ce6-Leu@Mn ²⁺ probe was investigated using the method of oxygen generation in living systems. Photoacoustic imaging (PA), as a combination of optical and ultrasonic technologies, is an excellent imaging method with deep tissue penetration and high spatial resolution. PA imaging is used to detect oxygenated hemoglobin (HbO ₂ ) and probes in PA signals at 850 nm and 680 nm to monitor blood oxygen saturation changes within the tumor. Figures 9a and 9b show that mice injected with Ce6-Leu or Ce6-Ac@Mn ²⁺ (200 µL, 200 µM) recorded a weak PA signal of HbO ₂ (red, 850 nm) at the tumor site, and mice injected with Ce6-Leu@Mn ^{2 +} (200 µL, 200 µM) mice showed a significant increase in PA signal over time and reached a maximum at 6 hours (2.52 times that of Ce6-Leu). Furthermore, a similar trend of probe PA signal enhancement was observed at 680 nm (Fig. 10), suggesting that the probe could efficiently accumulate in the tumor area. Then, mouse tumors with different treatments (PBS, Ce6-Leu, Ce6-Ac@Mn ²⁺ , Ce6-Leu@Mn ²⁺ ) were dissected, and the sections were stained with anti-HIF-1α antibody. It was found that the elevated level of HIF-1α in the tumor tissues of Ce6-Leu@Mn ²⁺ mice was significantly reduced compared with other control groups (Fig. 9c and Fig. 11).

To further study the enhanced radiotherapy performance of the Ce6-Leu@Mn ²⁺ probe in vivo, BALB/c nude mice bearing HepG2 subcutaneous tumors were randomly divided into five groups (n=5) and received different treatments: PBS, X-ray Irradiation (RT), Ce6-Leu+X-ray irradiation (Ce6-Leu+RT), Ce6-Leu-Ac@Mn ²⁺ + rays (Ce6-Ac@Mn ²⁺ +RT) and Ce6-Leu@Mn ²⁺ +X-ray (Ce6-Leu@Mn ²⁺ +RT). The probe was injected into tumor-bearing mice through the tail vein for 6 hours, and the tumor was then irradiated with X-rays (8 Gy). The average tumor size of different groups of mice was monitored in real time over 14 days. As shown in Figures 9d and 12, the tumor growth trends were similar in the RT and Ce6-Leu+RT groups, with a reduction in tumor size of 22.8% and 18.6%, respectively, compared with the PBS group. The effect of Ce6-Ac@Mn ²⁺ +RT on tumor growth in mice was lower than that of the experimental group, only reaching a tumor inhibition rate of about 42.9%. In sharp contrast, Ce6-Leu@Mn ² The tumor size could be significantly reduced in ⁺ +RT, and the tumor growth inhibition rate was about 82%, compared with the other four control groups (Fig. 9e and Fig. 13a). Meanwhile, all these combined treatments had no significant effect on the body weight of mice (Fig. 13b), indicating that the probe Ce6-Leu@Mn ²⁺ has good biocompatibility in vivo. In order to further verify the radiotherapy effect, tumor tissues were extracted 48 hours after X-ray irradiation, followed by H&E and TUNEL staining. As shown in Figure 9f, severe nuclear pyknosis, apoptosis, and necrosis were detected in Ce6-Leu@Mn ²⁺ +RT (group V), and no obvious necrosis was seen in other control groups. Taken together, all these evidences highly demonstrate that the LAP/GSH-driven switchable therapeutic probe of the present invention can effectively improve tumor hypoxia and radiation therapy in vivo.

In order to overcome the shortcomings of traditional molecular probes for diagnosis and treatment, a tumor microenvironment-responsive near-infrared molecular probe was constructed, and the overexpressed leucine aminopeptidase and glutathione in tumor cells were used to trigger condensation reactions and then reassemble , so that the specific recovery of the ability of the probe to fluoresce and generate ROS at the tumor site, thereby effectively improving the imaging and treatment effect of the tumor. It has the following advantages: first, the condensation reaction is efficient, mild, fast, and highly selective; second, when the probe enters tumor cells, the overexpressed leucine aminopeptidase and glutathione Under the stimulation of glycine, the original amino group and sulfhydryl group in the cysteine structure are exposed, so that a click condensation reaction occurs, which is not affected by the external environment. The development of smart and shape-switchable nanomaterials that can undergo stimuli-responsive size switching in space and time holds great promise for improved tumor penetration and efficient drug delivery in vivo. The Mn ²⁺ chelating probe (Ce6-Leu@Mn ²⁺ ) was demonstrated to have the ability to catalyze the sustained generation of O ₂ from endogenous H ₂ O ₂ at hypoxic tumor sites, thereby improving oxygen supply to enhance radiotherapy efficacy. Therefore, the LAP/GSH-driven size-switchable nanosystem disclosed in the present invention will provide a new advanced technology to improve drug delivery efficiency for precise tumor diagnosis and treatment.

Claims (10)

1. A kind of intelligent conversion dual stimulation response type probe of chelating metal ions, it is characterized in that, the intelligent conversion dual stimulation response type probe of described chelation metal ion has following chemical structural formula:

   

   .
2. The application of the intelligent conversion dual stimulus-responsive probe for chelating metal ions according to claim 1 in the preparation of reagents for tumor diagnosis and/or treatment.
3. The use according to claim 2, characterized in that the treatment is radiotherapy.
4. The preparation method of the intelligent conversion double stimulus response type probe of the chelated metal ion of claim 1 is characterized in that, Ce6-Leu, inorganic manganese salt are mixed in solvent, add organic additive, stir to obtain the chelated metal ion Smart switching of dual stimulus-responsive probes.
5. According to claim 4, the preparation method of the intelligent conversion dual-stimuli-responsive probe for chelating metal ions is characterized in that compound 1 is subjected to amide condensation reaction with _NH2 -CBT to obtain compound 2; compound 2 is removed from the protecting group to obtain compound 3; compound 3 and N-fluorenylmethoxycarbonyl-S-tert-butylthio-L-cysteine carry out amide condensation reaction to obtain compound 4; compound 4 removes the protecting group to obtain compound 5; compound 5 and photosensitizer Reaction to obtain compound 6; compound 6 removes the protecting group to obtain compound 7; compound 7 undergoes amide condensation reaction with N-tert-butoxycarbonyl-L-leucine to obtain compound 8; compound 8 removes the protecting group to obtain Ce6-Leu.
6. The preparation method according to claim 5, wherein the molar ratio of compound 1 to NH ₂ -CBT is 1:1.2; the molar ratio of compound 5 to NHS-activated photosensitizer is 1.1:1; compound 3 and N- The molar ratio of fluorenylmethoxycarbonyl-S-tert-butylthio-L-cysteine is 1:1.2; the molar ratio of compound 7 to N-tert-butoxycarbonyl-L-leucine is 1:1.2.
7. The preparation method according to claim 5, characterized in that the photosensitizer is chlorin E6.
8. The preparation method according to claim 5, characterized in that the inorganic manganese salt is manganese chloride, the solvent is methanol, and the organic additive is pyridine.
9. The preparation method according to claim 5, characterized in that the stirring is performed at 35-40° C. for 3-5 hours.
10. The application of the intelligent conversion dual stimulus-responsive probe for chelating metal ions according to claim 1 in the preparation of synergistic reagents for tumor radiotherapy.