Preparation method and application of near-infrared two-zone ratio type nitric oxide probe

Abstract

The invention discloses a preparation method and application of a near-infrared two-region ratio type nitric oxide probe, and relates to the technical field of biological medicines. The method comprises the following steps: uniformly dispersing lead sulfide nanoparticles and conjugated polymer fluorescent molecules responsive to nitric oxide in chloroform, simultaneously adding a polymer for improving particle dispersity, mixing and reacting, and purifying a reaction product to obtain the near-infrared two-zone ratio-type nitric oxide probe. The ratio type probe is prepared by combining lead sulfide quantum dots (the emission wavelength is 1500-1700 nm) with a nitric oxide response type conjugated polymer (the emission wavelength is 900-1200 nm after the nitric oxide response type conjugated polymer reacts with NO). The probe can smoothly enter cells, and the concentration of nitric oxide in the cells can be judged by measuring a fluorescence signal ratio by using a near-infrared two-region imaging platform.

Description

Preparation method and application of a near-infrared II ratiometric nitric oxide probe Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for preparing and applying a near-infrared II ratiometric nitric oxide probe. Background Technology

[0002] The discovery of gaseous signaling molecules is one of the most remarkable scientific achievements of the past few decades. For example, nitric oxide, carbon monoxide, and hydrogen sulfide, once considered toxic gaseous molecules, have now been proven to participate in various physiological and pathophysiological processes. Among them, the star molecule nitric oxide has been shown to play important roles in endothelial vasodilation, cardiovascular function regulation, antibacterial effects, wound healing, tissue repair, neurotransmitter transmission, and immune regulation. For instance, a decrease in nitric oxide and impaired release can lead to atherosclerosis and coronary artery spasm; while an increase in nitric oxide synthesis can easily cause hypotension, even hemorrhagic and anaphylactic shock, in patients with liver dysfunction. Furthermore, nitric oxide is a poorly water-soluble and unstable free radical molecule, readily oxidized to nitrite or nitrate in biological systems, making direct in vivo measurement of nitric oxide relatively difficult. Therefore, achieving real-time, accurate, and visual detection of in vivo nitric oxide levels is of profound significance for a deeper understanding of its biological functions and the diagnosis and treatment of nitric oxide-related diseases.

[0003] While near-infrared I (700-900 nm) fluorescence imaging is widely used in non-invasive biological observation, its shallow tissue penetration depth and strong biological tissue scattering and autofluorescence interference limit the signal-to-noise ratio and quantitative accuracy. Near-infrared II (1000-1700 nm) imaging, with its lower tissue scattering and autofluorescence background, significantly improves imaging penetration depth, spatial resolution, and signal-to-noise ratio, making it an ideal technology for achieving high-fidelity imaging of deep tissues. However, translating these advantages into high-precision dynamic monitoring of specific biomolecules (such as nitric oxide) still faces key challenges: most reported NIR-II nitric oxide probes are currently designed as "on" single-channel probes, whose fluorescence signals are easily interfered with by non-specific factors such as probe local concentration distribution, instrument parameters, and microenvironmental changes, making accurate quantification in vivo difficult. To address this issue, ratiometric fluorescence imaging strategies can effectively correct the aforementioned interference by simultaneously detecting signals at two different wavelengths and calculating their ratio, providing a feasible path for reliable quantification of nitric oxide in vivo. However, the application of this strategy in the NIR-II window is still relatively limited, and existing probes still have limitations in terms of photostability, water solubility, and signal-to-noise ratio, which restricts its further biological applications.

[0004] Based on this, the present invention provides a ratiometric near-infrared II nitric oxide probe, which, compared with other nitric oxide probes, can achieve highly sensitive visualization and quantitative analysis of nitric oxide in vivo, and can be used as an effective analytical tool for studying nitric oxide-related physiological and pathological processes. Summary of the Invention

[0005] To address the shortcomings of the aforementioned background technology, this invention provides a method for preparing and applying a near-infrared II ratiometric nitric oxide probe. This method combines lead sulfide quantum dots (emission wavelength at 1500-1700 nm) with a nitric oxide-responsive conjugated polymer (emission wavelength at 900-1200 nm after reacting with NO) to prepare a ratiometric probe. This probe can readily enter cells, and the intracellular nitric oxide concentration can be determined by measuring the fluorescence signal ratio using a near-infrared II imaging platform. Furthermore, in models such as skin inflammation, this ratiometric probe can accurately detect the nitric oxide content in in vivo inflamed areas.

[0006] The first objective of this invention is to provide a method for preparing a near-infrared II ratiometric nitric oxide probe, comprising the following steps: uniformly dispersing lead sulfide nanoparticles and nitric oxide-responsive conjugated polymer fluorescent molecules in chloroform; simultaneously, adding a polymer to improve particle dispersibility; after mixing and reacting, purifying the reaction product to obtain the near-infrared II ratiometric nitric oxide probe; wherein the nitric oxide-responsive conjugated polymer fluorescent molecules are prepared according to the following steps: dissolving 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole and 2,7-di(trimethyltin)-4,4,9,9-tetra(p-hexylbenzene)-indargen[1,2-b:5,6-b']dithiophene in toluene; under the action of a catalyst, after a first degassing and heating reaction, obtaining an intermediate product; dissolving the intermediate product and iron powder in a mixed solvent of acetic acid and chloroform; after a second degassing and heating reaction, obtaining the nitric oxide-responsive conjugated polymer fluorescent molecules.

[0007] Preferably, the lead sulfide nanoparticles are prepared according to the following steps: lead chloride is dissolved in oleylamine, sulfur is added in an anaerobic environment, the mixture is reacted, and then a mixed solution of n-hexane and anhydrous ethanol is added to terminate the reaction, thereby obtaining lead sulfide nanoparticles.

[0008] Preferably, the lead sulfide nanoparticles have a particle size of 5-10 nm.

[0009] Preferably, the reaction temperature for preparing lead sulfide nanoparticles is 155-165℃; and the molar ratio of lead chloride to sulfur is 1:0.1-0.5.

[0010] Preferably, the molecular weight ratio of the lead sulfide nanoparticles to the nitric oxide-responsive conjugated polymer fluorescent molecules is 1:2 to 3:1.

[0011] Preferably, the polymer used to improve particle dispersibility is oleylamine-grafted 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)], or oleylamine-grafted polyacrylic acid; the mass ratio of the polymer used to improve particle dispersibility to the lead sulfide nanoparticles is 1~5:1.

[0012] Preferably, the reaction temperature after the first degassing is 105-115℃, and the reaction time is 12-24h; the reaction temperature after the second degassing is 75-85℃, and the reaction time is 2-4h.

[0013] Preferably, after purifying the reaction product, the method further includes dispersing the purified product in a phosphate buffer solution to obtain a ratiometric probe working solution of appropriate concentration.

[0014] The second objective of this invention is to provide a near-infrared II ratiometric nitric oxide probe.

[0015] The third objective of this invention is to provide an application of a near-infrared II ratiometric nitric oxide probe in a device for detecting nitric oxide content in in vivo inflammatory areas.

[0016] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention provides a method for preparing and applying a near-infrared II ratiometric nitric oxide probe. Based on the fact that nitric oxide is a key signaling molecule playing an important role in various physiological activities, this invention detects changes in nitric oxide concentration in organisms under different physiological activities and during different disease processes, which is of great significance for a deeper understanding of life activities and disease mechanisms. Currently, probes capable of highly sensitively detecting nitric oxide concentration in vivo are rarely reported. This invention discovers that nitric oxide-responsive conjugated polymers can be prepared through simple chemical synthesis, which promotes the development of polymer-based fluorescent probe tools.

[0017] This invention utilizes a simple method to bind a nitric oxide-responsive conjugated polymer to the surface of lead sulfide quantum dots, fabricating a novel near-infrared II ratiometric nitric oxide probe. This probe not only rapidly detects nitric oxide concentration in solution but also, due to the high penetration and low noise imaging advantages of the near-infrared II region, enables real-time detection of nitric oxide signals in vivo. Furthermore, as a ratiometric nitric oxide probe, it allows for quantitative analysis of nitric oxide concentration at disease sites. This probe provides a powerful tool for monitoring nitric oxide-related physiological activities, particularly for the diagnosis of inflammation-related diseases. Simultaneously, the design and fabrication strategy of this ratiometric probe provides theoretical support and reference for the development of detection systems for other physiological signaling molecules.

[0018] This invention, by combining conjugated polymer fluorescent molecules on the surface of lead sulfide quantum dots, can simultaneously modify polyacrylic acid grafted with 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]] or oleylamine, which has long blood circulation function, thereby improving the circulation stability of ratiometric probes in vivo and effectively reducing their recognition and clearance by the immune system, thus achieving higher detection efficiency and imaging effect. This design can provide ideas for the design of in vivo diagnostic systems.

[0019] This invention employs a simple and efficient preparation and modification method to prepare a near-infrared II ratiometric nitric oxide detection probe, achieving high sensitivity, high resolution, and low noise in vivo nitric oxide detection. The probe exhibits excellent imaging performance and quantitative analysis capabilities at the cellular level and in various inflammatory models. The preparation process of this ratiometric nitric oxide probe can be scaled up for production, which is of great significance for promoting the clinical translation and widespread application of in vivo real-time fluorescent probes. Attached Figure Description

[0020] Figure 1 is a schematic diagram of the synthesis of near-infrared II conjugated polymers that respond to nitric oxide. P1 is the conjugated polymer before reacting with nitric oxide, and P2 is the conjugated polymer after reacting with nitric oxide.

[0021] Figure 2 shows the NMR spectrum (a) and gel permeation chromatography (GPC) chromatogram (b) of the near-infrared two-region conjugated polymer in response to nitric oxide.

[0022] Figure 3 is a transmission electron microscope image of a near-infrared II ratiometric nitric oxide fluorescent probe.

[0023] Figure 4 shows the UV absorption curve of the near-infrared II ratiometric nitric oxide fluorescent probe (CSQD@PIB) as a function of nitric oxide concentration.

[0024] Figure 5 shows the variation of near-infrared fluorescence signal of the near-infrared II ratiometric nitric oxide fluorescent probe (CSQD@PIB) with nitric oxide concentration.

[0025] Figure 6 shows fluorescence imaging images of the near-infrared II ratiometric nitric oxide fluorescent probe (CSQD@PIB) as the nitric oxide concentration increases. From left to right, the images are: white light image, near-infrared IIa region image, near-infrared IIb region image, and IIa / IIb region overlap image.

[0026] Figure 7 shows the fluorescence image and corresponding signal intensity statistics of the near-infrared II ratiometric nitric oxide fluorescent probe (CSQD@PIB) in macrophages for detecting nitric oxide concentration.

[0027] Figure 8 shows the in vivo fluorescence imaging of nitric oxide detected by the near-infrared II ratiometric nitric oxide fluorescent probe (CSQD@PIB) in a mouse skin inflammation model.

[0028] Figure 9 shows the fluorescence imaging of skin tissue sections in a mouse skin inflammation model using the infrared two-zone ratiometric nitric oxide fluorescent probe (CSQD@PIB) to detect nitric oxide.

[0029] Figure 10. In vivo fluorescence imaging of nitric oxide detected by the near-infrared II ratiometric nitric oxide fluorescent probe (CSQD@PIB) in a mouse colitis model.

[0030] Figure 11 is a statistical graph of nitric oxide signals in the colitis region of Figure 10.

[0031] Figure 12 Fluorescence imaging images of mouse colon tissue with different degrees of colitis using a near-infrared II ratiometric nitric oxide fluorescent probe (CSQD@PIB), where the stronger the inflammation, the brighter the red. Detailed Implementation

[0032] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention.

[0033] This invention employs a ratiometric fluorescence imaging strategy to simultaneously detect two signals of different wavelengths and calculate their ratio, effectively correcting the aforementioned interference and providing a feasible path for reliable quantification of nitric oxide in vivo. However, the application of this strategy in the NIR-II window is still relatively limited, and existing probes still have limitations in terms of photostability, water solubility, and signal-to-noise ratio, which restricts their further biological applications.

[0034] The purpose of this invention is to provide a near-infrared II ratiometric fluorescent probe with high sensitivity and specificity. A ratiometric probe is prepared by combining lead sulfide quantum dots (emission wavelength at 1500-1700 nm) and a nitric oxide-responsive conjugated polymer (emission wavelength at 900-1200 nm after reacting with NO). This probe can readily enter cells, and the intracellular nitric oxide concentration can be determined by measuring the fluorescence signal ratio using a near-infrared II imaging platform. Furthermore, in models such as skin inflammation, this ratiometric probe can accurately detect the nitric oxide content in in vivo inflamed areas.

[0035] To achieve the above objectives, the first aspect of the present invention provides a method for preparing a near-infrared II ratiometric nitric oxide probe, comprising the following steps: uniformly dispersing lead sulfide nanoparticles and nitric oxide-responsive conjugated polymer fluorescent molecules in chloroform; simultaneously, adding a polymer to improve particle dispersibility; after mixing and reacting, purifying the reaction product to obtain the near-infrared II ratiometric nitric oxide probe; wherein the mass ratio of the lead sulfide nanoparticles to the nitric oxide-responsive conjugated polymer fluorescent molecules is 1:2 to 3:1, preferably 5:2.

[0036] The polymer used to improve particle dispersibility is oleylamine-grafted 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] or oleylamine-grafted polyacrylic acid, both purchased from Maclean.

[0037] The mass ratio of the polymer used to improve particle dispersibility to the lead sulfide nanoparticles is 1 to 5:1, preferably 3:1.

[0038] After purifying the reaction product, the process also includes dispersing the purified product in a phosphate buffer solution to obtain a ratiometric probe working solution of appropriate concentration.

[0039] This invention uses a simple method to bind nitric oxide-responsive conjugated polymer fluorescent molecules to the surface of lead sulfide quantum dots, thus preparing a novel near-infrared II ratiometric nitric oxide probe. This probe can not only rapidly detect nitric oxide concentration in solution, but also detect nitric oxide signals in vivo in real time due to the high penetration and low noise imaging advantages of the near-infrared II region.

[0040] The ratio of lead sulfide nanoparticles to conjugated polymer fluorescent molecules in a near-infrared II ratiometric nitric oxide probe can be adjusted to regulate the system's response characteristics and signal intensity to nitric oxide.

[0041] Polymers that improve the dispersibility of infrared II ratiometric nitric oxide probes are polymers with good in vivo circulation effects, including polymers that prolong in vivo circulation, such as oleylamine-grafted polyacrylic acid, 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)], and targeting ligands that enhance the enrichment effect in lesion areas.

[0042] Targeting ligand molecules are tumor cell-specific targeting molecules, including antibodies, membrane proteins, peptides, polysaccharides, etc.

[0043] The nitric oxide-responsive conjugated polymer fluorescent molecule was prepared according to the following steps: 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole and 2,7-di(trimethyltin)-4,4,9,9-tetra(p-hexylbenzene)-indargeno[1,2-b:5,6-b']dithiophene were dissolved in toluene. Under the action of a catalyst, the mixture underwent a first degassing reaction followed by heating to obtain an intermediate product. The intermediate product and iron powder were dissolved in a mixed solvent of acetic acid and chloroform. After a second degassing reaction, the mixture was heated to obtain the nitric oxide-responsive conjugated polymer fluorescent molecule. The iron powder was used to reduce the nitro groups in the molecule to amino groups.

[0044] After the first degassing, the reaction temperature is 105-115℃ and the reaction time is 12-24h; after the second degassing, the reaction temperature is 75-85℃ and the reaction time is 2-4h.

[0045] In a mixed solvent of acetic acid and chloroform, the volume ratio of acetic acid to chloroform is 1:1.

[0046] The lead sulfide nanoparticles are prepared according to the following steps: lead chloride is dissolved in oleylamine, sulfur is added in an anaerobic environment, the mixture is reacted, and then a mixed solution of n-hexane and anhydrous ethanol is added to terminate the reaction, thus obtaining lead sulfide nanoparticles.

[0047] The lead sulfide nanoparticles have a particle size of 5-10 nm; their particle size can be controlled by changing the amount of lead chloride and sulfur added.

[0048] When preparing lead sulfide nanoparticles, the reaction temperature is 155-165℃; the molar ratio of lead chloride to sulfur is 1:0.1-0.5, preferably 1:0.25.

[0049] After the reaction is terminated, lead sulfide particles are collected by centrifugation and stored in n-hexane.

[0050] A second aspect of the present invention provides a near-infrared II ratiometric nitric oxide probe.

[0051] The third aspect of this invention provides the application of a near-infrared II ratiometric nitric oxide probe in a device for detecting nitric oxide content in in vivo inflammatory areas.

[0052] This invention utilizes an innovative chemical reaction pathway to combine near-infrared IIb region (emission wavelength: 1500-1700 nm) quantum dot lead sulfide with a nitric oxide-specific responsive conjugated polymer probe in the near-infrared IIb region (emission wavelength: 900-1200 nm) to prepare a novel near-infrared II region ratiometric nitric oxide probe. This method is simple and low-cost. Further modification with polymers that prolong in vivo circulation and targeting ligands results in a ratiometric probe with a uniform particle size distribution. Its lead sulfide core exhibits stable and clear near-infrared IIb region imaging capabilities, while the surface-bound conjugated polymer fluorescent molecules are highly responsive to nitric oxide, with the fluorescence signal increasing with increasing nitric oxide concentration. By calculating the ratio, the nitric oxide concentration in in vivo lesion areas can be accurately and quantitatively analyzed.

[0053] It should be noted that, unless otherwise specified, the experimental methods used in this invention are all conventional methods; and the reagents and materials used, unless otherwise specified, are all commercially available.

[0054] Example 1 This example describes the preparation method and in vitro nitric oxide detection verification of a near-infrared II ratiometric nitric oxide fluorescent probe (CSQD@PIB). The main steps include: (1) Synthesis and characterization of nitric oxide-responsive near-infrared II conjugated polymer fluorescent molecules. As shown in Figure 1, 247 mg of M1 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole, 77 mg of M2 2,7-di(trimethyltin)-4,4,9,9-tetra(p-hexylphenyl)-indargen[1,2-b:5,6-b']dithiophene and 15 mg of tetra(triphenylphosphine)palladium were dissolved in 15 mL of toluene. The mixture was degassed three times, heated to 110 °C, and stirred for 24 hours. After cooling to room temperature, 100 mL of methanol was added. The precipitated polymer was washed with n-hexane and dried under vacuum to obtain P1, with a yield of approximately 57%. The above product and 84 mg of iron powder were dissolved in 16 mL of a 1:1 volume ratio mixture of ethanol and chloroform. After degassing, the mixture was reacted at 80 °C for 4 hours. After cooling to room temperature, the product was extracted with dichloromethane, dried over magnesium sulfate, and concentrated. Methanol was added to the liquid, and the precipitate was the desired conjugated polymer, with a yield of approximately 78%. Referring to Figure 2, the NMR results (a) and gel permeation chromatography (GPC) results (b) show that the chemical structure is as expected. GPC indicates that the conjugated polymer has a molecular weight of 59,500 g / mol, a PDI of 1.81, and a narrow molecular weight distribution.

[0055] (2) Preparation of lead sulfide nanoparticles. Lead chloride (834 mg, 3 mmol) was dissolved in oleylamine (7.5 mL) and degassed at 120 °C for 30 min. The reaction flask was then filled with nitrogen and heated to 160 °C. Next, a solution of sulfur (24 mg, 0.75 mmol) dissolved in oleylamine (2.25 mL) was added to the above mixture. After reacting for 30 min, the reaction was terminated by adding a mixed solution of n-hexane and anhydrous ethanol (30 mL, volume ratio 1:2). The lead sulfide nanoparticles were collected by centrifugation and resuspended in n-hexane (10 mL) for storage.

[0056] (3) Preparation of near-infrared II ratiometric nitric oxide fluorescent probe (CSQD@PIB). 15 mg of oleylamine-grafted polyacrylic acid was dissolved in 2 mL of chloroform. Then, 5 mg of the prepared lead sulfide nanoparticles (CSQD) and 2 mg of nitric oxide conjugated polymer probe (PIB) were added to the solution, and the mixture was stirred continuously at room temperature for 30 minutes. The organic solvent was then removed by rotary evaporation. 2 mL of sodium carbonate aqueous solution (0.5%, w / v) was added to the product, and the solution was sonicated to ensure complete dissolution. The solution was then purified by ultrafiltration to remove unreacted raw materials and residual organic solvent. Finally, the purified product was redispersed in 5 mL of phosphate buffered saline to obtain a CSQD@PIB dispersion with a concentration of 1 mg / mL for storage. As shown in Figure 3, transmission electron microscopy images show that the prepared CSQD@PIB probe has a uniform and regular morphology with a particle size of approximately 8 nm. In Figure 3, (a) and (b) are transmission electron microscope images of lead sulfide particles, and (c) and (d) are CSQD@PIB particles.

[0057] (4) Verification of the nitric oxide detection capability of the near-infrared II ratiometric fluorescent probe (CSQD@PIB) in solution. Probe solutions containing different nitric oxide concentrations were detected using a UV-Vis spectrophotometer and a near-infrared II fluorescence spectrophotometer. Figure 4 shows the UV-Vis absorption spectrum; with increasing NO concentration, the UV-Vis absorption spectrum of the probe exhibits a regular red shift. Figure 5 illustrates that the fluorescence intensity in the 900–1200 nm channel (IIa, NO response channel) increases in a concentration-dependent manner, while the fluorescence intensity in the 1500–1700 nm channel (IIb, reference channel) remains essentially unchanged. Further verification of the probe's responsiveness to nitric oxide was performed using a near-infrared II imaging system. As shown in Figure 6, the acquired fluorescence images show that with increasing nitric oxide concentration, the blue fluorescence of the conjugated polymer probe gradually increases, while the fluorescence of lead sulfide remains essentially unchanged. The ratio of the two increases linearly with increasing nitric oxide concentration, indicating that the CSQD@PIB has a sensitive nitric oxide detection function in solution.

[0058] (5) Raw264.7 cells were incubated with 0.2 mg / mL CSQD@PIB probe for 30 minutes, and near-infrared dual-channel (IIa and IIb) imaging was performed under 660 nm excitation light, as shown in Figure 7. The imaging results showed that in the IIb channel (internal reference signal), the fluorescence signal intensity of the experimental group and the control group was similar; while in the IIa channel (NO response signal), compared with the unstimulated control group, the fluorescence signal of cells treated with LPS for 2 hours and 6 hours showed a gradient increase. Quantitative analysis of the fluorescence intensity ratio (FIIa / FIIb) in the region of interest (ROI) revealed that the fluorescence ratio of the LPS-stimulated group for 2 hours and 6 hours increased significantly compared with the control group. This NIR-II imaging result indicates that the CSQD@PIB probe can effectively monitor the M1 polarization process of Raw264.7 macrophages through its specific response to NO.

[0059] Example 2 This example uses the near-infrared II ratiometric nitric oxide fluorescent probe (CSQD@PIB) prepared in Example 1 for the detection of skin inflammation.

[0060] (1) Validation of the performance of the near-infrared II ratiometric nitric oxide fluorescent probe (CSQD@PIB) in real-time in vivo detection of nitric oxide in a mouse skin inflammation model. Mouse skin inflammation was induced with LPS. Six hours after subcutaneous injection of LPS, an equal amount of the near-infrared II CSQD fluorescent probe was subcutaneously injected at the same site. In vivo NIR-II fluorescence imaging was performed at 10, 30, and 60 minutes post-injection using 660 nm laser excitation. The probe signal was clearly observed on the mouse back at all time points (see Figure 8). Quantitative analysis of the intensity ratio of the two fluorescence channels (FIIa / FIIb) within the region of interest (ROI) revealed a significant increasing fluorescence ratio on the LPS-inflamed side compared to the control side over time. This indicates that the probe can be used for real-time detection of nitric oxide during skin inflammation.

[0061] (2) The near-infrared II ratiometric nitric oxide fluorescent probe (CSQD@PIB) was used to detect nitric oxide concentration in tissue sections of a mouse skin inflammation model. After in vivo imaging, mice were sacrificed and local skin tissue was collected for further analysis. First, in vitro NIR-II imaging was performed on the skin sections, as shown in Figure 9. The fluorescence ratio was calculated at the cellular level. The results showed that the trend of the ratio change was consistent with the in vivo imaging results, proving that the probe CSQD@PIB can sensitively detect nitric oxide levels in tissue sections and even in vivo.

[0062] Example 3: This example demonstrates the use of the near-infrared II ratiometric nitric oxide fluorescent probe (CSQD@PIB) prepared in Example 1 for the detection of colonic inflammation.

[0063] (1) Validation of the performance of near-infrared II ratiometric nitric oxide fluorescent probe (CSQD@PIB) in real-time in vivo detection of nitric oxide in a mouse model of colonic inflammation. Different degrees of colonic inflammation were induced with different concentrations of DSS. On day 7 of induction, all mice were given 200 μL of 1 mg / mL CSQD@PIB probe solution via enema, and in vivo near-infrared II (NIR-II) fluorescence imaging was performed at 10, 30, and 60 minutes. Imaging showed obvious fluorescence signals in the mouse abdomen and clear intestinal contours, demonstrating the high signal-to-noise ratio advantage of NIR-II imaging (see Figure 10). Quantitative analysis of the fluorescence ratio (FIIa / FIIb) showed significant differences among the three groups (see Figure 11), confirming that CSQD@PIB can non-invasively detect and quantify nitric oxide (NO) levels in the colon.

[0064] (2) Near-infrared II ratiometric nitric oxide fluorescent probe (CSQD@PIB) was used to detect nitric oxide concentration in tissue sections of a mouse colonic inflammation model. Mice were sacrificed after in vivo imaging, and the colon was harvested for in vitro imaging and histological analysis. As shown in Figure 12, the imaging results of the in vitro sections showed that the fluorescence ratio increased with increasing DSS concentration, consistent with the in vivo observation results. Spatial heterogeneity analysis was performed on the colon of mice in the 2.5% DSS group. Based on the NIR-II imaging of the in vitro colon, segments with high fluorescence ratios (Severe segment) and low fluorescence ratios (Medium segment) were extracted for section analysis. NIR-II fluorescence imaging at the section level showed that the fluorescence ratio of the Severe segment was significantly higher than that of the Medium segment. H&E staining and immunofluorescence (CD86 / iNOS) analysis of this segment also confirmed that the Severe segment exhibited more severe tissue damage and stronger inflammatory signals, achieving the correlation verification between macroscopic imaging and microscopic pathology. The spatial heterogeneity analysis described above demonstrates that the CSQD@PIB probe not only non-invasively detects overall inflammatory changes at the organ level but also possesses excellent spatial resolution, accurately revealing the microscopic heterogeneity of inflammation distribution within tissues. Its fluorescence signal intensity exhibits highly quantitative spatial co-localization with the severity of local pathology, confirming the probe's significant potential for precise quantification and visualization of disease activity focal points, surpassing traditional detection methods that only provide overall average information.

[0065] Example 4 is the same as Example 1, except that the molecular weight ratio of lead sulfide nanoparticles to the nitric oxide-responsive conjugated polymer fluorescent molecules is 1:2; the molecular weight ratio of the polymer used to improve particle dispersibility to the lead sulfide nanoparticles is 1:1; and the molar ratio of lead chloride to sulfur is 1:0.1.

[0066] Example 5 is the same as Example 1, except that the molecular weight ratio of lead sulfide nanoparticles to the nitric oxide-responsive conjugated polymer fluorescent molecules is 3:1; the molecular weight ratio of the polymer used to improve particle dispersibility to the lead sulfide nanoparticles is 5:1; and the molar ratio of lead chloride to sulfur is 1:0.5.

[0067] This invention describes preferred embodiments and their effects. However, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments.

[0068] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a near-infrared II ratiometric nitric oxide probe, characterized in that, Includes the following steps: Lead sulfide nanoparticles and nitric oxide-responsive conjugated polymer fluorescent molecules were uniformly dispersed in chloroform. Simultaneously, a polymer was added to improve particle dispersibility. After mixing and reacting, the reaction product was purified to obtain a near-infrared II ratiometric nitric oxide probe. The nitric oxide-responsive conjugated polymer fluorescent molecules were prepared according to the following steps: 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole and 2,7-di(trimethyltin)-4,4,9,9-tetra(p-hexylbenzene)-indargen[1,2-b:5,6-b']dithiophene were dissolved in toluene. Under the action of a catalyst, the mixture underwent a first degassing reaction followed by heating to obtain an intermediate product. The intermediate product and iron powder were dissolved in a mixed solvent of acetic acid and chloroform, and after a second degassing reaction, the mixture was heated to obtain the nitric oxide-responsive conjugated polymer fluorescent molecules.

2. The method for preparing a near-infrared II ratiometric nitric oxide probe according to claim 1, characterized in that, The lead sulfide nanoparticles are prepared according to the following steps: lead chloride is dissolved in oleylamine, sulfur is added in an anaerobic environment, the mixture is reacted, and then a mixed solution of n-hexane and anhydrous ethanol is added to terminate the reaction, thus obtaining lead sulfide nanoparticles.

3. The method for preparing a near-infrared II ratiometric nitric oxide probe according to claim 2, characterized in that, The lead sulfide nanoparticles have a particle size of 5-10 nm.

4. The method for preparing a near-infrared II ratiometric nitric oxide probe according to claim 2, characterized in that, When preparing lead sulfide nanoparticles, the reaction temperature is 155-165℃; the molar ratio of lead chloride to sulfur is 1:0.1-0.

5.

5. The method for preparing a near-infrared II ratiometric nitric oxide probe according to claim 1, characterized in that, The molecular weight ratio of the lead sulfide nanoparticles to the nitric oxide-responsive conjugated polymer fluorescent molecules is 1:2 to 3:

1.

6. The method for preparing a near-infrared II ratiometric nitric oxide probe according to claim 1, characterized in that, The polymer used to improve particle dispersibility is oleylamine-grafted 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)], or oleylamine-grafted polyacrylic acid; the mass ratio of the polymer used to improve particle dispersibility to the lead sulfide nanoparticles is 1~5:

1.

7. The method for preparing a near-infrared II ratiometric nitric oxide probe according to claim 1, characterized in that, After the first degassing, the reaction temperature is 105-115℃ and the reaction time is 12-24h; after the second degassing, the reaction temperature is 75-85℃ and the reaction time is 2-4h.

8. The method for preparing a near-infrared II ratiometric nitric oxide probe according to claim 1, characterized in that, After purifying the reaction product, the process also includes dispersing the purified product in a phosphate buffer solution to obtain a ratiometric probe working solution of appropriate concentration.

9. A near-infrared II ratiometric nitric oxide probe prepared by the method of any one of claims 1 to 8.

10. The application of the near-infrared two-zone ratiometric nitric oxide probe of claim 9 in a device for detecting nitric oxide content in in vivo inflammatory areas.

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