Ratio probe for accurate diagnosis and treatment of radioactive intestinal injury and preparation and application thereof

Abstract

The invention discloses a ratio probe for precise diagnosis and treatment of radioactive intestinal injury as well as a preparation method and application thereof, relates to the technical field of nano biological materials, and solves the technical problems that in the prior art, radiation enteritis diagnosis depends on subjective symptoms and invasive examination, intestinal ROS cannot be monitored noninvasively and quantitatively, and RIE is difficult to diagnose early and evaluate dynamically. The ratio probe for accurate diagnosis and treatment of radioactive intestinal injury is prepared, and the ratio probe comprises a rare earth doped down-conversion nanoparticle core-shell structure DCNP and has near-infrared second-region NIR-II emission capability; the reactive oxygen species (ROS)-sensitive IR786s dye molecules are coated on the surface of the nanoparticle core-shell structure DCNP, and are used as a response unit of the ROS and a regulation and control unit of an absorption competition induced emission (ACIE) mechanism; a bacterial membrane EM of Escherichia coli Nissle 1917EcN is coated on the outer side of a coating of a nanoparticle core-shell structure DCNP for modifying IR786s dye, so that bionic camouflage of the nanoprobe is realized, the residence time of the nanoprobe in an intestinal tract is prolonged, and the targeting property of the nanoprobe to a diseased region is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of nanobiomaterials, and specifically to a ratio probe for precise diagnosis and treatment of radiation-induced intestinal injury, and its preparation and application. Background Art

[0002] Currently, the diagnosis of radiation-induced enteritis (RIE) mainly relies on the subjective symptom description of patients and invasive endoscopic examinations. However, the subjective symptom assessment lacks objectivity and accuracy, making it difficult to detect and accurately evaluate the severity of the disease at an early stage; although endoscopic examinations can provide intuitive morphological information of the intestinal mucosa and histopathological evidence, they are invasive and have certain risks such as bleeding and perforation, and are also costly, with poor patient compliance, and are not suitable for early screening, frequent monitoring, and long-term follow-up of RIE.

[0003] Therefore, there is an urgent clinical need for a non-invasive, sensitive, accurate, quantifiable, and real-time monitoring imaging technology for the ROS level in the intestine to achieve early diagnosis of RIE, monitoring of disease progression, and evaluation of treatment effects. Summary of the Invention

[0004] In view of the deficiencies and drawbacks in the prior art, the present invention provides a ratio probe for precise diagnosis and treatment of radiation-induced intestinal injury, which can achieve real-time, quantitative, and non-invasive monitoring of ROS in radiation-induced intestinal injury (RIE), and can be used to evaluate treatment effects, thereby realizing precise diagnosis and treatment monitoring of radiation-induced intestinal injury, as well as its preparation and application.

[0005] To achieve the above objectives, the present invention is realized through the following technical solutions: The ratio probe for precise diagnosis and treatment of radiation-induced intestinal injury provided by the present invention includes a fluorescence signal generation unit, a reactive oxygen species (ROS) response unit, and a targeting unit that are connected to each other;

[0006] The core fluorescence signal generation unit is a rare earth-doped down-conversion nanoparticle core-shell structure (DCNP) with the ability to emit in the second near-infrared region (NIR-II), where the core component is Er 3+ / Yb 3+ co-doped, and the shell component is Nd 3+ / Y 3+ co-doped;

[0007] The middle layer ROS response unit is a ROS-sensitive IR786s dye molecule coating wrapped on the surface of DCNP to obtain DCNP@IR786s. Through the absorption competition-induced emission (ACIE) mechanism, that is, by detecting the change in the ratio of NIR-II emission intensities at different excitation wavelengths, ratio-type fluorescence sensing of ROS is achieved;

[0008] The outermost targeting unit is the bacterial membrane (EM) of Escherichia coli Nissle 1917 EcN, which is coated on the outside of the DCNP coating modified with IR786s dye to prepare the DCNP@786s@EM nanoprobe, realizing the biomimetic camouflage of the nanoprobe and improving its retention time in the intestine and targeting to the lesion site.

[0009] Preferably, the DCNP core-shell structured nanoparticles generate a first NIR-II emission peak under 808 nm laser excitation and a second NIR-II emission peak under 980 nm laser excitation, and the intensity ratio of the first NIR-II emission peak to the second NIR-II emission peak is used to reflect the ROS level.

[0010] Preferably, the particle size of DCNP is 40 nm - 50 nm.

[0011] Preferably, the IR786s dye molecule coating realizes the ratio-type fluorescence sensing of ROS through the absorption competition induced emission (ACIE) mechanism, and specifically reflects the ROS level by detecting the change in the ratio of the NIR-II emission intensities under 808 nm excitation and 980 nm excitation.

[0012] The preparation method of the ratio probe for precise diagnosis and treatment of radiation intestinal injury according to any one of the above includes the following steps:

[0013] S1. Synthesize DCNP by the thermal decomposition method, including the following steps:

[0014] S1.1 Synthesize the NaYbF4:2%Er, 2%Ce core: Add YbCl3·6H2O, ErCl3·6H2O, CeCl3·6H2O, oleic acid and octadecene into a three-necked flask according to a certain proportion. Under N2 protection, heat to 160 °C and keep the temperature constant until the reaction is complete, then cool down; then add the methanol solution dissolved in NaOH and NH4F, stir at room temperature, and then successively raise the temperature to 70 °C for reaction for 15 min, raise the temperature to 100 °C for reaction for 30 min, and finally raise the temperature to 300 °C for reaction for 60 min, naturally cool, centrifuge, wash with n-hexane, and ultrasonically disperse to obtain the core;

[0015] S1.2 Coating the NaYF4:30%Nd shell: Dissolve the above core in hexane, add oleic acid and octadecene, and then add Y(CF3COO)3, Nd(CF3COO)3 and sodium oleate; first heat to 120 °C, then raise the temperature to 310 °C and keep it for 60 min, centrifuge, wash with n-hexane, and ultrasonically disperse to obtain the nanoparticle core-shell structure DCNP;

[0016] S2) Modify the ROS-sensitive IR786s dye molecule on the surface of DCNP to prepare the nano-composite system DCNP@IR786s;

[0017] S3) Coating the outer side of the DCNP@IR786s with EM to obtain the ratio-type rare earth fluorescent nanoprobe DCNP@786s@EM.

[0018] Preferably, the molar ratio of NH4F to rare earth ions is 4:1; the molar ratio of NaOH to NH4F is 5:8; the volume ratio of oleic acid to octadecene is 2:5;

[0019] The molar ratio of Y(CF3COO)3, Nd(CF3COO)3 to sodium oleate is 7:3:20.

[0020] Preferably, in step S2, IR786s and DSPE-PEG2000 are coated on the surface of DCNP by the thin film hydration self-assembly method to obtain DCNP@IR786s, including the following specific steps:

[0021] Dissolve DCNP, DSPE-PEG2000 and IR786s in chloroform, wherein the mass ratio of DCNP to DSPE-PEG2000 is 1:3, and the mass ratio of DCNP to IR786s is 25:1, stir overnight at room temperature;

[0022] After rotary evaporation to form a film, redissolve with deionized water, and purify by centrifugation to obtain DCNP@IR786s.

[0023] Preferably, the steps of extracting the bacterial membrane (EM) of Escherichia coli Nissle 1917 and coating EM on the outer side of DCNP@IR786s by the extrusion method to obtain the DCNP@786s@EM nanoprobe include the following specific steps:

[0024] Culture Escherichia coli Nissle 1917;

[0025] Collect the bacterial cells, and lyse the bacteria with lysozyme, Triton X-100, MgCl2 and DNase;

[0026] Obtain the bacterial membrane by differential centrifugation and ultracentrifugation;

[0027] Mix the bacterial membrane with DCNP@IR786s according to the mass ratio, and use an extruder to pass through porous polycarbonate membranes with pore sizes of 800 nm, 400 nm and 200 nm in sequence.

[0028] Preferably, it further includes the specific steps of synthesizing IR786s: dissolving IR775 chloride in DMF, adding 3-mercaptopropionic acid and triethylamine, stirring at room temperature, and performing post-treatment after the reaction.

[0029] Application of the ratio probe for precise diagnosis and treatment of radiation intestinal injury in the preparation of a reagent for non-invasive diagnosis or treatment monitoring of radiation enteritis.

[0030] Use of a ratio probe for precise diagnosis and treatment of radiation-induced intestinal injury in the preparation of a reagent for detecting or quantifying ROS in a biological sample or living tissue.

[0031] Use of a ratio probe for precise diagnosis and treatment of radiation-induced intestinal injury in the preparation of a reagent for evaluating the therapeutic effect of radiation enteritis.

[0032] The ratio probe for precise diagnosis and treatment of radiation-induced intestinal injury provided by the present invention, its preparation and application have the following beneficial effects:

[0033] (1) The ratio probe for precise diagnosis and treatment of radiation-induced intestinal injury of the present invention has the following advantages:

[0034] High sensitivity and specificity: Based on the absorption competition-induced emission (ACIE) mechanism, a dual-wavelength excitation strategy of 808 nm and 980 nm is adopted to achieve ratio-type fluorescence detection of reactive oxygen species (ROS). Compared with traditional single-wavelength fluorescence probes, the present invention can sensitively reflect the dynamic changes of ROS levels through the change of ratio fluorescence signals, effectively eliminate background interference, improve signal contrast, make the detection results more accurate and reliable, and is particularly suitable for the detection of low-concentration ROS, providing a highly specific index for the early diagnosis of radiation enteritis.

[0035] Non-invasive and real-time: The ratio-type rare-earth fluorescence nanosensor DCNP@786s@EM probe in the present invention has near-infrared second window (NIR-II) fluorescence emission characteristics, can penetrate deep tissues and reduce the interference of autofluorescence of biological tissues, and is suitable for non-invasive in vivo imaging. Through NIR-II fluorescence imaging technology, the development process of radiation enteritis can be monitored in real time and dynamically, effectively avoiding the invasive operation risks of traditional endoscopy, significantly improving patient compliance, and is particularly suitable for long-term efficacy evaluation and disease tracking.

[0036] Targeting and long-acting: The surface of the ratio-type rare-earth fluorescence nanosensor DCNP@786s@EM of the present invention is coated with Escherichia coli bacterial membrane EM for biomimetic camouflage, endowing it with excellent biocompatibility and biomimetic intestinal colonization ability, and being able to simulate the intestinal colonization characteristics of bacteria, improving the retention time of the probe in the intestine and the targeting to the lesion site, extending the effective monitoring time, that is, it can actively target the intestinal injury and inflammation site and extend the intestinal retention time to ensure long-term effective monitoring.

[0037] Diagnosis and treatment integration: The nanosensor of the present invention can not only be used for the early diagnosis and disease progression monitoring of radiation enteritis, but also can not only accurately diagnose the severity of radiation enteritis, but also evaluate the effect of antioxidant therapy by real-time monitoring the change of ROS level, such as drug responsiveness monitoring and efficacy prediction, so as to guide the adjustment of individualized treatment plans and truly realize the integrated application of "diagnosis-treatment-evaluation". Brief Description of the Drawings

[0038] Figure 1 Schematic diagram of the synthesis and characterization of the DCNP@786s nanoprobe in the present invention; (A) Schematic diagram of the preparation of the DCNP@786s nanoprobe. (B) Transmission electron microscope (TEM) image of DCNP@786s. Scale bar: 200 nm. (C) Element distribution map of the prepared core-shell structure. Scale bar: 100 nm. (D) Hydrodynamic particle size distribution of DCNP@786s. (E) Particle size stability of DCNP@786s in PBS (pH = 7.4, black line) and 10% FBS (red line). (F) UV-visible absorption spectra of DCNP, IR786s, and DCNP@786s. (G) Fourier transform infrared (FTIR) spectra of DCNP, IR786s, and DCNP@786s. (H) NIR-II fluorescence signals of DCNP and DCNP@786s under 808 nm laser excitation. (I) NIR-II fluorescence signals of DCNP and DCNP@786s under 980 nm laser excitation.

[0039] Figure 2 Spectrum diagram of ROS-induced NIR-II fluorescence regulation of DCNP@786s in the present invention; (A) Schematic diagram showing the energy transfer process in the designed core-shell nanostructure. NIR-II spectra of DCNP@786s after treatment with different ROS: ONOO - (B), ·OH (C), ClO - (D), H2O2 (E), and O2 ·- (F), obtained under 808 nm laser excitation. (G) Spectra obtained under 980 nm laser excitation. (H) Sensitivity of DCNP@786s to different types of ROS. (I) NIR-II imaging of DCNP@786s after treatment with different types of ROS.

[0040] Figure 3This is the in vitro verification result graph of the ratio of NIR-II imaging and DCNP@786s in monitoring radiation-induced cytotoxicity and oxidative stress in the present invention; (A) Flow cytometry analysis of ROS production in NCM460 cells after X-ray irradiation, using APF staining. (B) Cytotoxicity of X-ray irradiation on HCT-116 tumor spheroids. Live cells and dead cells were stained with Calcein-AM and PI, respectively. (C) Relative fluorescence intensity in NCM460 cells after X-ray irradiation, using APF staining. (D) Analysis of relative fluorescence intensity of live cells and dead cells. (E) Western blot analysis of protein expression in NCM460 cells after X-ray irradiation. (F) NIR-II fluorescence imaging of DCNP@786s-labeled NCM460 cells treated with different doses of X-ray irradiation for 2 days.

[0041] Figure 4 This is the quantitative analysis graph of the enhanced intestinal retention ability of DCNP@786s by EM coating in the present invention; (A) Schematic diagram of the preparation of DCNP@786s@EM. (B) TEM image of DCNP@786s@EM. (C) Zeta potential of DCNP, DCNP@786s and DCNP@786s@EM. (D) Ex vivo fluorescence imaging of major organs (heart, liver, spleen, lung, kidney and colon) of mice after oral administration of DCNP@786s@EM for 48 h. (E) Ex vivo NIR-II fluorescence imaging of the gastrointestinal tract of mice after oral administration of DCNP@786s (left) and DCNP@786s@EM (right) for 48 h. (F) Quantitative analysis of gastrointestinal fluorescence intensity.

[0042] Figure 5 This is the in vivo diagnostic analysis graph of DCNP@786s@EM for the RIE mouse model in the present invention; (A) Schematic diagram of the establishment of the RIE mouse model and non-invasive in vivo diagnosis. (B) NIR-II fluorescence (FL) images (excited at 808 nm and 980 nm) of radioactive enteritis mice after oral administration of DCNP@786s@EM at 1 h, 2 h, 4 h and 8 h after irradiation with different X-ray doses (0 Gy, 6 Gy, 12 Gy). (C) Relative fluorescence intensity under 808 nm excitation. (D) Relative fluorescence intensity under 980 nm excitation; (E) Ratio of fluorescence intensity at 1450 nm under 808 nm excitation and 980 nm excitation (F 1450,808Ex / F 1450,980Ex )

[0043] Figure 6This is a real-time monitoring diagram of the therapeutic effect of DCNP@786s@EM in the present invention on the RIE mouse model; (A) Schematic diagram of in vivo ratiometric NIR-II FL imaging for the monitoring of radiation enteritis treatment; (B) After oral administration of DCNP@786s@EM, the F 1550,808Ex / F 1550,980Ex ratiometric NIR-II FL images of mice with radiation enteritis at different time points (D0, D3, D6, D9, D12) (G1: healthy control group; G2: 6Gy X-ray irradiation group; G3: 12Gy X-ray irradiation group; G4: 6Gy X-ray irradiation + dexamethasone treatment group; G5: 12Gy X-ray irradiation + dexamethasone treatment group); (C-G) Individual data of the F 1550,808Ex / F 1550,980Ex ratio of each group of mice; (H) Summary diagram.

[0044] Figure 7 This is a comprehensive evaluation diagram of the therapeutic effect of DCNP@786s@EM in the present invention on the RIE mouse model. (A) H&E staining analysis of small intestine and colon tissues. (B) Representative images of the colon. (C) Quantification of colon length. (D) Histological score. (E) Body weight. (F) Percentage of survival rate. (G) Disease activity index. Levels of different inflammatory factors in colon tissues, including IL-1β (H), IL-6 (I), IL-12 (J), TNF-α (K). Detailed implementation mode

[0045] The technical solutions of the present invention are described in detail below through exemplary specific embodiments. However, these embodiments should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0046] Unless otherwise specified, the raw materials and reagents described in the embodiments are all commercially available products.

[0047] Example 1

[0048] The ratiometric probe for precise diagnosis and treatment of radiation intestinal injury of the present invention includes a fluorescence signal generation unit, a reactive oxygen species (ROS) response unit, and a targeting unit connected to each other;

[0049] The core fluorescence signal generation unit is a rare earth doped down-conversion nanoparticle core-shell structure (DCNP) with near-infrared second region (NIR-II) emission ability. Among them, the core component is Er 3+ / Yb 3+ co-doped, and the shell component is Nd 3+ / Y 3+Co-doping; The DCNP core-shell structured nanoparticles generate a first NIR-II emission peak under 808 nm laser excitation and a second NIR-II emission peak under 980 nm laser excitation. The intensity ratio of the first NIR-II emission peak to the second NIR-II emission peak is used to reflect the ROS level. The particle size of DCNP is 40 nm - 50 nm.

[0050] The middle layer ROS-responsive unit is a ROS-sensitive IR786s dye molecule coating wrapped on the surface of DCNP to obtain DCNP@IR786s. Through the absorption competition-induced emission (ACIE) mechanism, that is, by detecting the change in the ratio of NIR-II emission intensities at different excitation wavelengths, the ratio-type fluorescence sensing of ROS is realized; the R786s dye molecule coating realizes the ratio-type fluorescence sensing of ROS through the absorption competition-induced emission (ACIE) mechanism, specifically by detecting the change in the ratio of NIR-II emission intensities under 808 nm excitation and 980 nm excitation to reflect the ROS level.

[0051] The outermost layer targeting unit is the bacterial membrane (EM) of Escherichia coli Nissle 1917 EcN, which is coated on the outside of the DCNP coating modified with IR786s dye to obtain the DCNP@786s@EM nanoprobe.

[0052] Example 2

[0053] Preparation of DCNP@786s nanoprobe and characterization of its ROS-responsive NIR-II fluorescence properties.

[0054] It aims to verify the ability of DCNP@786s@EM to detect ROS at the cellular level, the effect of X-ray irradiation on cells, and evaluate the application of DCNP@786s@EM as a ratio-type NIR-II fluorescence probe in cell imaging.

[0055] As Figure 1 shown in A, in this example, the DCNP@786s nanoprobe is prepared by the step-by-step growth method and the self-assembly method. The process is as Figure 1 shown in A, including the following specific steps:

[0056] S1. Synthesize DCNP by the thermal decomposition method, including the following steps:

[0057] S1.1 Synthesis of NaYbF4:2% Er, 2% Ce core: 0.96 mmol of YbCl3·6H2O, 0.02 mmol of ErCl3·6H2O, 0.02 mmol of CeCl3·7H2O, 6 ml of oleic acid and 15 ml of octadecene were added to a three-necked flask in a certain ratio and mixed. Under N2 protection, the temperature was raised from room temperature to 160 °C within 50 min and kept at a constant temperature for 60 min until the reaction was complete, and then cooled to below 40 °C. A 10 ml methanol solution containing 2.5 mmol of NaOH (0.1 g) and 4 mmol of NH4F (0.148 g) was added, stirred at room temperature, and then heated to 70 °C for 15 min, then heated to 100 °C for 30 min, and finally heated to 300 °C for 60 min, followed by natural cooling, centrifugation, washing with n-hexane, and ultrasonic dispersion to obtain the core.

[0058] S1.2 Coating of NaYF4:30% Nd shell: The above core was dissolved in hexane, oleic acid and octadecene were added, heated to 120 °C and kept for 30 min. 0.7 mmol of Y(CF3COO)3, 0.3 mmol of Nd(CF3COO)3 and 2 mmol of sodium oleate solution were added dropwise, then heated to 310 °C and kept for 60 min, centrifuged, washed with n-hexane, and ultrasonically dispersed to obtain the nanoparticle core-shell structure DCNP. The molar ratio of Y(CF3COO)3, Nd(CF3COO)3 to sodium oleate is 7:3:20.

[0059] S2) Modifying the surface of DCNP with ROS-sensitive IR786s dye molecules to prepare the nanocomposite system DCNP@IR786s, including the following steps:

[0060] S21 Dissolve IR775 chloride in DMF, add 3-mercaptopropionic acid and triethylamine, stir at room temperature, and perform post-treatment after the reaction to obtain IR786s.

[0061] S22 Coating IR786s and DSPE-PEG2000 on the surface of DCNP by the thin film hydration self-assembly method to obtain DCNP@IR786s, including the following specific steps:

[0062] Dissolve DCNP, DSPE-PEG2000 and IR786s in chloroform, where the mass ratio of DCNP to DSPE-PEG2000 is 1:3, and the mass ratio of DCNP to IR786s is 25:1. Stir at room temperature overnight; after rotary evaporation to form a film (volatilize the solvent), redissolve with deionized water, centrifuge and purify to obtain DCNP@IR786s.

[0063] Material characterization: Transmission electron microscopy (TEM) was used to observe the morphology and size of the nanoprobe, as Figure 1As shown in Figure B, the results showed that DCNP@786s was in the shape of regular spheres with a clear core-shell structure and an average particle size of about 44.9 ± 2.3 nm. Elemental mapping confirmed the distribution of each element in the core-shell structure, as Figure 1 shown in Figure C.

[0064] Dynamic light scattering (DLS) measurements verified similar results, with a slightly increased average particle size, as Figure 1 shown in Figure D. The colloidal stability of DCNP@786s was evaluated by monitoring the particle size changes in phosphate-buffered saline (PBS, pH 7.4) and medium (DMEM) containing 10% fetal bovine serum (FBS), as Figure 1 shown in Figure E. The results showed that the particle size remained relatively stable, indicating that almost no aggregation or degradation occurred under biological conditions.

[0065] Ultraviolet-visible (UV-Vis) absorption spectroscopy confirmed that IR786s was successfully coated on DCNPs, and the drug loading efficiency of DCNP@786s was about 25%, as Figure 1 shown in Figure F.

[0066] To further confirm the modification of the surface of DCNPs by IR786s, Fourier transform infrared (FTIR) spectroscopy analysis was carried out. Characteristic absorption peaks appeared in the spectrum at approximately 1720 cm -1 (attributed to C=O stretching vibration), 1540 cm -1 and 1460 cm -1 (corresponding to aromatic ring stretching vibration) and a broad peak in the range of 1100 - 1200 cm -1 (related to C-O-C stretching vibration), as Figure 1 shown in Figure G.

[0067] Based on the ACIE mechanism, under 808 nm laser excitation, the near-infrared II (NIR-II) fluorescence intensity of DCNP@786s decreased significantly, as Figure 1 shown in Figure H. In contrast, no obvious change was observed in the spectrum under 980 nm laser excitation, indicating that IR786s was successfully coated on the surface of DCNP, as Figure 1 shown in Figure I.

[0068] As Figure 2 shown in Figure A, the DCNP@786s nanoprobe presented a core-shell structure, containing an Er 3+ / Yb 3+ core and a Nd 3+ / Y 3+ shell. Under 808 nm laser excitation, the down-conversion process was initiated within the shell, where Nd 3+ ions served as sensitizers, absorbing 808 nm photons and transferring the energy to Yb in the shell 3+ions. Subsequently, energy is transferred to the core, where Yb 3+ ions act as an energy bridge to transfer the excitation energy to Er 3+ activator ions. This cascading process ultimately results in the characteristic NIR-II emission of Er 3+ at approximately 1550 nm (4I 13 / 2 →4I 15 / 2 transition). In contrast, direct excitation of Yb 3+ ions in the core by a 980 nm laser also induces NIR-II emission, providing a basis for internal reference.

[0069] ONOO - ·OH, and ClO - induced a concentration-dependent increase in the NIR-II fluorescence intensity at 1550 nm under 808 nm excitation in the concentration range of 0 to 50 μM ( Figure 2 B-D). This indicates that these reactive oxygen species (ROS) weaken the competitive absorption of IR786s to 808 nm light, thereby enhancing the NIR-II emission. In contrast, the addition of HO or O ·- had little effect on the changes in the NIR-II fluorescence spectrum, indicating that it had little effect on the ACIE process, as shown in Figure 2 E and Figure 2 F. Notably, the fluorescence excited by 980 nm was not affected by any ROS, further confirming that the 808 nm excitation emission was specifically regulated by ROS through the ACIE mechanism.

[0070] To further clarify the optical response mechanism of DCNP@786s, the ratio of the emission intensity at 1550 nm under 808 nm excitation to the emission intensity under 980 nm excitation was calculated. As shown in Figure 2 H, an obvious relationship was observed between this ratio and the ROS concentration, revealing the ROS-regulated ACIE process. In addition, the fluorescence imaging of DCNP@786s solution exposed to different ROS intuitively demonstrated the ROS response behavior of the nanoprobe, as shown in Figure 2 I.

[0071] Example 2

[0072] To evaluate the radiation-induced cell damage, normal human colon epithelial cell line (NCM460) was exposed to different doses of X-ray radiation (0 Gy, 2 Gy, 4 Gy, 6 Gy). Subsequently, the ROS level was evaluated using the 3'-(p-aminophenyl) fluorescein (APF) probe. The results of fluorescence imaging and flow cytometry both showed a dose-dependent increase in fluorescence intensity, indicating that a higher X-ray dose promoted the generation of ROS, as shown in Figure 3 A and Figure 3 C.

[0073] In addition, the effects of damage caused by X-ray radiation on HCT-116 tumor cell spheroids were further investigated, as Figure 3 shown in B. Calcein-AM / PI staining showed that as the X-ray dose increased from 2 Gy to 6 Gy, the Calcein-AM fluorescence gradually decreased. At the same time, the fluorescence intensity of PI staining increased, indicating a gradual loss of cell membrane integrity and an increase in cell death, as Figure 3 shown in D.

[0074] To further clarify the mechanism of X-ray-induced cytotoxicity, the present invention used Western blot analysis to detect the expression of γ-H2AX, a marker of DNA double-strand breaks. As Figure 3 shown in E, compared with the control group (0 Gy), X-ray radiation significantly increased the expression of γ-H2AX protein. These results strongly suggest that X-ray radiation-induced cytotoxicity is mediated by ROS-induced DNA damage, ultimately leading to cell death.

[0075] After X-ray radiation treatment, NIR-II fluorescence imaging was performed by exciting with 808 nm and 980 nm and collecting fluorescence through a 1450 nm long-pass filter. As Figure 3 shown in F, the fluorescence excited by 980 nm remained relatively consistent at all radiation doses, indicating that the DCNPs themselves were not significantly affected by the ROS generated during the radiation process. In contrast, the fluorescence intensity excited by 808 nm increased with increasing X-ray dose, suggesting that the increased oxidative stress may lead to the degradation of IR786s, thereby restoring the NIR-II emission of DCNPs. The ratio of the fluorescence intensity excited by 808 nm to that excited by 980 nm provides a quantitative measurement of ROS generation. These results indicate that DCNP@786s can effectively monitor radiation-induced cell damage in real time by tracking ROS generation.

[0076] Example 3

[0077] EM coating enhances the intestinal retention ability of DCNP@786s.

[0078] This example aims to endow DCNP@786s@EM with biomimetic properties through bacterial membrane coating technology and study its effect on the intestinal retention ability of the nanoprobe.

[0079] S3. Coat EM on the outer side of the coating of DCNP@IR786s to prepare the ratio-type rare earth fluorescent nanoprobe DCNP@786s@EM, including the following steps:

[0080] Step S31 for extracting the bacterial membrane (EM) of Escherichia coli Nissle 1917 and coating the EM outside DCNP@IR786s by extrusion to obtain the DCNP@786s@EM nanoprobe includes the following specific steps:

[0081] Culture Escherichia coli Nissle 1917;

[0082] Collect the bacterial cells and lyse the bacteria with lysozyme, Triton X-100, MgCl2 and DNase;

[0083] Obtain the bacterial membrane by differential centrifugation and ultracentrifugation;

[0084] Mix the bacterial membrane with DCNP@IR786s and use an extruder to pass through porous polycarbonate membranes with pore sizes of 800 nm, 400 nm and 200 nm in sequence.

[0085] Step S32 for coating EM outside the coating of DCNP@IR786s to prepare the ratio-type rare earth fluorescent nanoprobe DCNP@786s@EM includes the following specific steps:

[0086] As Figure 4 shown in A, coat the bacterial membrane (EM) of Escherichia coli Nissle 1917 (EcN 1917) on the surface of DCNP@786s by extrusion to obtain DCNP@786s@EM.

[0087] Use a transmission electron microscope (TEM) to observe the morphology of DCNP@786s@EM. As Figure 4 shown in B, DCNP@786s@EM presents a clear core-shell structure, and the nanoparticles are uniformly coated with a thin film, indicating that the bacterial membrane is successfully coated on the surface of DCNP@786s.

[0088] Use a dynamic light scattering instrument (DLS) to measure the Zeta potential of DCNP, DCNP@786s and DCNP@786s@EM. As Figure 4 shown in C, the Zeta potential of DCNP is +4.1 mV, while the Zeta potential of DCNP@786s@EM becomes -11.2 mV, indicating that the negatively charged bacterial membrane is successfully coated on the surface of the nanoprobe.

[0089] To evaluate the in vivo intestinal retention ability of DCNP@786s@EM, DCNP@786s and DCNP@786s@EM were respectively administered to BALB / c mice by oral gavage. 48 h after administration, a small animal in vivo imaging system was used to observe the distribution of the nanoprobe in the mice. The mice were sacrificed, and the main organs (heart, liver, spleen, lung, kidney) and gastrointestinal tract were taken out for ex vivo NIR-II fluorescence imaging. The results are asFigure 4 As shown in D, no obvious fluorescence signal was detected in the major organs of mice 8 h after oral administration of DCNP@786s@EM, while strong NIR-II fluorescence signal was detected in the colon region. The gastrointestinal tract of the mice was further dissected for ex vivo imaging analysis, and the results are as Figure 4 shown in E. Obvious NIR-II fluorescence signal remained in the gastrointestinal tract of the mice in the DCNP@786s@EM group 48 h after oral administration, while the fluorescence signal in the gastrointestinal tract of the mice in the DCNP@786s group was very weak.

[0090] Quantitative analysis was performed on the gastrointestinal fluorescence signal ( Figure 4 F). The fluorescence intensity of the DCNP@786s@EM group was significantly higher than that of the DCNP@786s group (about 2.1 times). The above results indicate that the DCNP@786s@EM nanoprobe prepared by the bacterial membrane coating technology can effectively mimic the characteristics of bacteria, significantly increase its intestinal retention time in mice, and achieve targeted enrichment in the colon, thereby prolonging the observation time.

[0091] Example 4

[0092] This example aims to use the DCNP@786s@EM nanoprobe for the diagnosis, treatment effect monitoring and comprehensive efficacy evaluation of radiation-induced enteritis (RIE) in vivo.

[0093] After one-week adaptive feeding of 6-8-week-old BALB / c mice, they were irradiated with different doses of X-rays (0, 6, 12 Gy) using a small animal irradiator (RS2000Pro-225, Rad Source, U.S.) to establish an RIE mouse model.

[0094] As Figure 5 shown in A, at different time points (1 h, 2 h, 4 h, 8 h) after X-ray irradiation, DCNP@786s@EM (7.5 mg / kg) was administered to the mice in each group by oral gavage. NIR-II fluorescence images were collected using a small animal in vivo imaging system (PerkinElmer IVIS Lumina) under excitation at 808 nm and 980 nm respectively, and the ratio of fluorescence intensity at 1450 nm (F 1550,808Ex / F 1550,980Ex ) was calculated to achieve ratio imaging and quantitative analysis of the ROS level in the intestine. During the treatment process (D0, D3, D6, D9, D12), NIR-II fluorescence imaging was repeated to monitor the changes in the ROS level.

[0095] As Figure 5As shown in Figure B, under 808 nm excitation, the fluorescence signal in the intestinal region of RIE mice was significantly stronger than that in the healthy control group, and the fluorescence intensity increased with the increase of irradiation dose; under 980 nm excitation, there was no significant difference in the fluorescence intensity in the intestinal region of each group of mice. 1550,808Ex / F 1550,980Ex The ratio ( Figure 5 C-E) could more clearly distinguish different X-ray irradiation dose groups, indicating that DCNP@786s@EM could be used for the diagnosis of RIE in vivo and reflect the ROS level through ratio-type fluorescence signals.

[0096] As Figure 6 shown in Figure A, mice were randomly divided into the following groups: Group G1: healthy control group (n = 5); Group G2: 6 Gy X-ray irradiation group (n = 5); Group G3: 12 Gy X-ray irradiation group (n = 5); Group G4: 6 Gy X-ray irradiation + dexamethasone treatment group (n = 5); Group G5: 12 Gy X-ray irradiation + dexamethasone treatment group (n = 5). As Figure 6 shown in Figure B, with the progress of dexamethasone treatment, the F 1550,808Ex / F 1550,980Ex ratio in the intestines of the treated mice (G4, G5) gradually decreased, indicating a decrease in ROS level; while the F 1550,808Ex / F 1550,980Ex ratio in the intestines of the untreated mice (G2, G3) remained at a high level, indicating a continuous increase in ROS level. Figure 6 Figures C-H show the change curves of the F 1550,808Ex / F 1550,980Ex ratio of each group of mice. The results showed that DCNP@786s@EM could be used to monitor the treatment effect of RIE in real time.

[0097] As Figure 7 shown, the treated mice had less weight loss, increased survival rate, decreased DAI score, longer colon length, reduced histopathological damage, and lower levels of inflammatory factors, indicating that dexamethasone treatment effectively alleviated the symptoms and pathological damage of RIE. The results of blood routine and blood biochemical analysis showed that there were no obvious abnormalities in the main organ functions of each group of mice, indicating that both DCNP@786s@EM and dexamethasone treatment had good safety.

[0098] This example shows that the DCNP@786s@EM nanoprobe can be used for the early diagnosis, disease progression monitoring and treatment effect evaluation of RIE in vivo. Through ratio-type NIR-II fluorescence imaging, the changes in ROS level in the intestine can be monitored non-invasively, in real time and quantitatively, providing a promising tool for the precise diagnosis and treatment of RIE.

[0099] In summary, the DCNP@786s@EM nanoprobes of the present invention can be used for the early diagnosis, disease progression monitoring, and treatment effect evaluation of RIE in vivo. Through ratio-type NIR-II fluorescence imaging, the changes in ROS levels in the intestine can be monitored non-invasively, in real-time, and quantitatively, providing a promising tool for the precise diagnosis and treatment of RIE.

[0100] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A ratio probe for precise diagnosis and treatment of radiation-induced intestinal injury, characterized in that, It includes a mutually connected fluorescence signal generation unit and a reactive oxygen species (ROS) response unit; The core component of the fluorescence signal generation unit is a core-shell structure of rare-earth-doped down-conversion nanoparticles (DCNP) with the ability to emit in the second near-infrared region (NIR-II), where the core component is Er 3+ / Yb 3+ co-doped, and the shell component is Nd 3+ / Y 3+ co-doped; The intermediate layer ROS response unit is a ROS-sensitive IR786s dye molecule coating wrapped on the surface of DCNP to obtain DCNP@IR786s. Through the absorption competition-induced emission (ACIE) mechanism, that is, by detecting the change in the ratio of NIR-II emission intensities at different excitation wavelengths, the ratio-type fluorescence sensing of ROS is achieved.

2. The ratio probe for precise diagnosis and treatment of radioactive intestinal injury according to claim 1, wherein It also includes a targeting unit: the outermost targeting unit is the bacterial membrane (EM) of Escherichia coli Nissle 1917 EcN, which is coated on the outside of the DCNP coating modified with IR786s dye to obtain the DCNP@786s@EM nanoprobe.

3. The ratio probe for precise diagnosis and treatment of radioactive intestinal injury according to claim 1, characterized in that The DCNP core-shell structure nanoparticles generate a first NIR-II emission peak under 808 nm laser excitation and a second NIR-II emission peak under 980 nm laser excitation. The intensity ratio of the first NIR-II emission peak and the second NIR-II emission peak is used to reflect the ROS level; The particle size of the DCNP is 40 nm - 50 nm.

4. The ratio probe for precise diagnosis and treatment of radioactive intestinal injury according to claim 3, wherein The IR786s dye molecule coating realizes the ratio-type fluorescence sensing of ROS through the absorption competition-induced emission (ACIE) mechanism. Specifically, the change in the ratio of NIR-II emission intensities under 808 nm excitation and 980 nm excitation is detected to reflect the ROS level.

5. The preparation method of the ratio probe for precise diagnosis and treatment of radioactive intestinal injury according to any one of claims 1-4, characterized in that, It includes the following steps: S1. Synthesize DCNP by the thermal decomposition method, including the following steps: S1.1 Synthesize the NaYbF4:2%Er, 2%Ce core: Add YbCl3·6H2O, ErCl3·6H2O, CeCl3·6H2O, oleic acid and octadecene into a three-necked flask according to a certain ratio. Under N2 protection, heat to 160 °C and keep the temperature constant until the reaction is complete, then cool down; then add the methanol solution dissolved in NaOH and NH4F, stir at room temperature, and then raise the temperature to 70 °C for 15 min, raise the temperature to 100 °C for reaction, and finally raise the temperature to 300 °C for 60 min. Naturally cool, centrifuge and wash with n-hexane, and ultrasonically disperse to obtain the core; S1.2 Coating the NaYF4:30%Nd shell: Dissolve the above core in hexane, add oleic acid and octadecene, and then add Y(CF3COO)3, Nd(CF3COO)3 and sodium oleate; first heat to 120 °C, then raise the temperature to 310 °C and keep it for 60 min, centrifuge and wash with n-hexane, and ultrasonically disperse to obtain the nanoparticle core-shell structure DCNP; S2. Modify the ROS-sensitive IR786s dye molecule on the surface of DCNP to obtain the nanocomposite system DCNP@IR786s; S3. Coating EM on the outside of the DCNP@IR786s coating to obtain the ratio-type rare earth fluorescence nanoprobe DCNP@786s@EM.

6. The preparation method of the ratio probe for precise diagnosis and treatment of radioactive intestinal injury according to claim 5, wherein, In step S1.1, the molar ratio of NH4F to rare earth ions is 4:1; the molar ratio of NaOH to NH4F is 5:8; the volume ratio of oleic acid to octadecene is 2:5; The molar ratio of Y(CF3COO)3, Nd(CF3COO)3 to sodium oleate is 7:3:

20.

7. The preparation method of the ratio probe for precise diagnosis and treatment of radioactive intestinal injury according to claim 5, characterized in that, In step S2, IR786s and DSPE-PEG2000 are coated on the surface of DCNP by the method of thin-film hydration self-assembly to obtain DCNP@IR786s, including the following specific steps: Dissolve DCNP, DSPE-PEG2000 and IR786s in chloroform, wherein the mass ratio of DCNP to DSPE-PEG2000 is 1:3, and the mass ratio of DCNP to IR786s is 25:1, and stir overnight at room temperature; After rotary evaporation to form a film, re-dissolve with deionized water and purify by centrifugation to obtain DCNP@IR786s.

8. The preparation method of the ratio probe for precise diagnosis and treatment of radioactive intestinal injury according to claim 5, characterized in that, The steps of extracting the bacterial membrane (EM) of Escherichia coli Nissle 1917 and coating the EM on the outside of DCNP@IR786s by the extrusion method to obtain the DCNP@786s@EM nanoprobe include the following specific steps: Culture Escherichia coli Nissle 1917; Collect the bacterial cells and lyse the bacteria with lysozyme, Triton X-100, MgCl2 and DNase; Obtain the bacterial membrane by differential centrifugation and ultracentrifugation; Mix the bacterial membrane with DCNP@IR786s and pass through porous polycarbonate membranes with pore sizes of 800 nm, 400 nm and 200 nm in sequence using an extruder.

9. The preparation method of the ratio probe for precise diagnosis and treatment of radiation-induced intestinal injury according to claim 5, characterized in that, It also includes the specific steps of synthesizing IR786s: dissolve IR775 chloride in DMF, add 3-mercaptopropionic acid and triethylamine, stir at room temperature, and perform post-treatment after the reaction to obtain it.

10. The application of the ratio probe for precise diagnosis and treatment of radiation-induced intestinal injury according to claim 1 in the preparation of a reagent for non-invasive diagnosis or treatment monitoring of radiation enteritis; or in the preparation of a reagent for detecting or quantifying ROS in a biological sample or living tissue; or in the preparation of a reagent for evaluating the treatment effect of radiation enteritis.

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