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WO2023098007A1 - Sonde sensible aux doubles stimuli à conversion intelligente chélatée avec des ions métalliques, son procédé de préparation et son utilisation - Google Patents

Sonde sensible aux doubles stimuli à conversion intelligente chélatée avec des ions métalliques, son procédé de préparation et son utilisation Download PDF

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WO2023098007A1
WO2023098007A1 PCT/CN2022/097205 CN2022097205W WO2023098007A1 WO 2023098007 A1 WO2023098007 A1 WO 2023098007A1 CN 2022097205 W CN2022097205 W CN 2022097205W WO 2023098007 A1 WO2023098007 A1 WO 2023098007A1
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compound
leu
probe
preparation
tumor
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史海斌
王安娜
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Suzhou University
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    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
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    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0036—Porphyrins
    • A—HUMAN NECESSITIES
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    • A61K49/0017—Fluorescence in vivo
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0089—Particulate, powder, adsorbate, bead, sphere
    • A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
    • A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/101—Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals
    • A61K49/106—Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals the complex-forming compound being cyclic, e.g. DOTA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
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    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • 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.
  • Rao and Liang's group innovatively proposed the concept of an enzyme/GSH-mediated self-assembly method based on biorthogonal CBT-Cys.
  • 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.
  • 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).
  • 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).
  • RT radiotherapy
  • an intelligent conversion dual-stimuli-response probe for chelating metal ions has the following chemical structural formula: .
  • 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
  • Ce6-Leu has the following chemical structural formula: .
  • 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.
  • 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.
  • 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.
  • 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.
  • step (5) the molar ratio of compound 5 to the photosensitizer is 1.1:1; the photosensitizer is NHS-activated chlorin E6 (Ce6-NHS).
  • 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.
  • 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.
  • 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.
  • 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.
  • the molar weight of the inorganic manganese salt is 4-6 times that of Ce6-Leu.
  • 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.
  • magnetic resonance imaging MRI
  • 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.
  • 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.
  • 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 ⁇ M, 200 ⁇ L) T1-weighted MR images with tumor aggregation and (g) Quantitative MR signal intensity changes in the tumor (I/I
  • Figure 4 is the characterization of the catalase-like probe Ce6-Leu@Mn 2+ ,
  • Figure 5 shows the toxicity of Ce6-Leu@Mn 2+ and Ce6-Ac@Mn 2+ to 3T3 cells.
  • FIG. 6 shows that the Ce6-Leu@Mn 2+ probe enhances the efficiency of radiotherapy in vitro
  • Scale bar 20 ⁇ m
  • (c) by ⁇ -H2AX To evaluate the DNA damage of HepG2 cells by different treatments.
  • Scale bar 20 ⁇ m
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 intermediate obtained by the group Fmoc reacts with N
  • hypoxia probe hypoxia red detection reagent
  • HIF-1 ⁇ levels were determined by western blot analysis. After 4 h incubation with different reagents (30 ⁇ M), HepG2 cells were washed 3 times with ice-cold PBS and lysed using RIPA lysis buffer containing complete protease inhibitors. 60 ⁇ g 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-1 ⁇ 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.
  • 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 ⁇ M) 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.
  • 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 ⁇ L of ⁇ -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.
  • Olympus confocal microscope Olympus, Tokyo, Japan
  • 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 ⁇ M) 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 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 ⁇ M, 200 ⁇ L). 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.
  • MOST real-time multispectral photoacoustic tomography system
  • Immunofluorescent staining was used to assess tumor hypoxia. Mice bearing HpeG2 tumors were randomly divided into 4 groups. After intravenous injection of PBS (200 ⁇ L), Ce6-Leu (200 ⁇ L, 200 ⁇ M), Ce6-Ac@Mn 2+ (200 ⁇ L, 200 ⁇ M) or Ce6-Leu@Mn 2+ (200 ⁇ L, 200 ⁇ M), tumors were dissected from mice and sectioned for Immunofluorescence staining for HIF-1 ⁇ was performed. Sections were observed with a confocal laser microscope.
  • 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 ⁇ m thick sections for hematoxylin-eosin (H&E) and TUNEL staining. Sections were then observed with a confocal laser microscope.
  • 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+ :
  • 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.
  • 2-amino-6-cyanobenzothiazole NH 2 -CBT, 179 mg,
  • 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.
  • 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).
  • Ce6-Leu@Mn 2+ for converting H 2 O 2 to O 2 in HepG2 cells was further evaluated.
  • 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.
  • HIF-1 ⁇ 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-1 ⁇ 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 ⁇ -H2AX immunofluorescent staining.
  • 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 ⁇ L, 200 ⁇ M) recorded a weak PA signal of HbO 2 (red, 850 nm) at the tumor site, and mice injected with Ce6-Leu@Mn 2 + (200 ⁇ L, 200 ⁇ M) 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.
  • mice with different treatments were dissected, and the sections were stained with anti-HIF-1 ⁇ antibody. It was found that the elevated level of HIF-1 ⁇ in the tumor tissues of Ce6-Leu@Mn 2+ mice was significantly reduced compared with other control groups (Fig. 9c and Fig. 11).
  • 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.
  • 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%.
  • 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.
  • 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.

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Abstract

L'invention concerne une sonde sensible aux doubles stimuli à conversion intelligente chélatée avec des ions métalliques, son procédé de préparation et son utilisation. La sonde peut être auto-assemblée en grosses nanoparticules dans une solution tampon d'eau, et les grosses nanoparticules sont ensuite converties en petites nanoparticules sous l'action de LAP et de GSH, de manière à renforcer leur accumulation dans une tumeur et leur pénétration dans un tissu profond, ce qui permet d'améliorer l'imagerie à proche infrarouge de la tumeur in vivo. De plus, il s'avère pour la première fois que ce procédé de désassemblage et de réduction de taille entraîné par LAP/GSH peut activer considérablement l'effet photodynamique d'un médicament thérapeutique, de manière à réaliser une thérapie photodynamique (PDT) efficace guidée par imagerie sur des tumeurs du foie et à réduire les effets secondaires sur les tissus normaux. La sonde de chélation Ce6-Leu@Mn 2+ peut améliorer l'alimentation en oxygène pour surmonter l'hypoxie et améliorer la génération de ROS sous radiation de rayons X, ce qui permet d'effectuer une radiothérapie efficace guidée par imagerie IRM sur des tumeurs HepG2 du foie humain chez des souris vivantes. Par conséquent, un nanosystème convertible en taille de la sonde de chélation peut fournir une technique puissante pour améliorer l'efficacité d'administration de médicament, ce qui permet d'améliorer le diagnostic et le traitement de tumeurs.
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