Preparation method of nitric oxide activated organic nano system and bimodal imaging application of nitric oxide activated organic nano system

Abstract

The invention discloses a preparation method of a nitric oxide (NO) activated organic nano system and bimodal imaging application of the nitric oxide (NO) activated organic nano system, and belongs to the technical field of biological imaging and biological pharmacy. The organic nano system (F NPs) is prepared from an optical functional probe FTEBD and an amphiphilic block copolymer PEG-b-PPG-b-PEG (F127) through a nano precipitation method. The NO activated organic nano system can specifically recognize NO in a microenvironment of an inflammation site and generate a detectable optical signal. The organic nano system can realize specific response in a mouse inflammation model, and provides an effective tool for accurate imaging of inflammation.

Description

Technical Field

[0001] This invention belongs to the field of bioimaging and biopharmaceutical technology, specifically relating to a method for preparing an organic nanosystem that can be used for NO response and its application in dual-modal imaging of inflammation. Background Technology

[0002] Inflammation is a self-protective response initiated by the body after tissues are damaged (such as by pathogens), often manifesting as redness, swelling, fever, and pain. As one of the most common pathological processes in human diseases, many illnesses are related to inflammation. Furthermore, inflammation caused by infectious pathogens is also contagious; for example, inflammation caused by Mycobacterium tuberculosis or influenza viruses can have a serious impact on socioeconomic life. In the examination of inflammation-related diseases, in addition to common examinations, doctors often use imaging techniques (such as X-rays, CT scans, and MRI) to locate the inflamed areas. To observe treatment effectiveness, these examinations may need to be performed multiple times, posing a potential hazard of ionizing radiation. Therefore, there is an urgent need to develop non-invasive, highly sensitive, high spatiotemporal resolution, and minimal side effects precise diagnostic methods to monitor changes in inflammation in real time.

[0003] Optical imaging technology, with its advantages of no ionizing radiation, low cost, real-time dynamic imaging, and non-invasive in vivo imaging, as well as its potential diffraction-limited or sub-diffraction-limited spatial resolution, plays a significant role in the fields of biology and medicine, becoming an important supplementary technology to X-ray computed tomography, magnetic resonance imaging, and ultrasound imaging. However, the spatial resolution and penetration depth of fluorescence imaging in living tissue are affected by a combination of factors, including light absorption, light scattering, tissue autofluorescence, probe quantum yield (QYs), optical structure design, and detector sensitivity and efficiency. Light scattering within tissue mainly consists of Rayleigh scattering and Mie scattering, the intensity of which decreases with increasing wavelength, following the principle of λ. α Therefore, reducing light scattering at longer wavelengths can achieve deeper penetration and higher contrast fluorescence imaging, thereby significantly reducing background signal interference and improving the imaging signal-to-noise ratio (SNR).

[0004] Photoacoustic imaging (PAI), a novel imaging modality complementary to optical imaging, has seen rapid development in recent years. PAI is a hybrid technique utilizing optical absorption and ultrasonic wave propagation, specifically the conversion between light and sound waves caused by the absorption of electromagnetic waves and localized thermal excitation. Therefore, as a non-invasive imaging method, PAI offers high contrast due to optical absorption, enabling selective excitation of spectral tissues with specific absorption characteristics, thus achieving functional imaging and obtaining molecular information. Furthermore, PAI detects ultrasonic waves generated after photothermal conversion, avoiding the influence of light scattering, thus possessing the high resolution and penetration depth of ultrasound imaging. However, compared to fluorescence imaging, PAI has lower sensitivity. Therefore, combining fluorescence imaging with PAI for multimodal imaging, through complementary advantages, can achieve both high imaging sensitivity and the acquisition of molecular information from deep tissues, improving the accuracy of imaging diagnosis and reducing the rates of missed and false diagnoses.

[0005] Nitric oxide (NO), a key regulatory molecule of inflammation, reflects the occurrence, development, and resolution of inflammation, and is crucial for disease progression. Elevated levels of nitric oxide synthase (iNOS) and NO are frequently detected in tissues of patients with rheumatoid arthritis, inflammatory bowel disease (IBD), and other conditions. Therefore, studying the spatiotemporal specific regulation of NO provides new targets for the treatment of inflammation-related diseases. Optical imaging, with its advantages such as ease of designing activation probes that specifically activate optical signals to biomarkers, has attracted widespread attention in the field of molecular imaging. Summary of the Invention

[0006] Based on this, the present invention aims to provide a NO-activated organic nanosystem, its preparation method, and its imaging application in inflammation. The material is prepared by nanoprecipitation of the biocompatible macromolecule F127 and the hydrophobic organic semiconductor probe FTEBD, resulting in a granular nanosystem.

[0007] This invention utilizes amphiphilic biomacromolecule self-assembly technology to construct a nanodelivery system with excellent biocompatibility, thereby improving the stability and biocompatibility of the material.

[0008] This invention discloses a method for preparing a NO-activated near-infrared II organic semiconductor fluorescent material, comprising the following steps: Step 1: Synthesis of organic semiconductor materials Utilizing the principle of flexible and processable organic semiconductors, a material with a D-π-A-π-D structure was designed. Using benzothiadiazole diamine as the weak acceptor unit, under NO stimulation, the thiadiazole diamine group transforms into a strong acceptor benzotriazole derivative, enhancing intramolecular charge transfer (ICT) and activating specific optical properties. Specific synthetic methods for the probe include the Suzuki-Miyaura coupling reaction, NBS bromination reaction, synthesis of tin reagents, and Stille coupling reaction. Finally, a reduction reaction is used to reduce the nitro group to an amino group, yielding the target probe FTEBD.

[0009] Step 2: Preparation of the nanosystem: The nanomaterial system was prepared using a nanoprecipitation method: Appropriate amounts of the probe and the biomacromolecule F127 (mass ratio 1:200) were weighed and dissolved completely in a suitable amount of THF solvent. The resulting solution was then injected into deionized water, repeatedly agitated, and finally evaporated completely using an N2 gas stream. Ultrafiltration using a 30 kDa ultrafiltration tube yielded the final nanomaterial mother liquor (F NPs).

[0010] This application provides a nitric oxide-activated organic nanosystem, comprising a photofunctional probe FTEBD and an amphiphilic block copolymer PEG- b -PPG- b -PEG was prepared into a nanosystem via nanoprecipitation. This activated organic nanosystem can specifically recognize NO in the microenvironment of inflammatory sites and generate a detectable optical signal. The structural formula of the optical functional probe FTEBD is as follows:

[0011] Formula I Where R1 is or R2 is or or .

[0012] This application provides a method for preparing the above-mentioned NO-activated organic nanosystem, comprising the following steps: Step 1: Fabrication of the optical functional probe FTEBD; Step 2: Weigh the probe FTEBD and the amphiphilic block copolymer F127 PEG- b -PPG- b -PEG was used to prepare NO-activated organic nano-systems F NPs via nanoprecipitation. The specific steps are as follows: Organic solvents were added to probes FTEBD and F127 to dissolve them, and the mixture was stirred to form a homogeneous solution. The solution was then rapidly injected into deionized water, repeatedly agitated, and sonicated to ensure uniform dispersion of the nanoparticles. Subsequently, N2 gas flow was used to agitate the liquid surface to completely remove THF. The solution was then concentrated by centrifugation using an ultrafiltration tube to obtain an aqueous dispersion mother liquor of F NPs.

[0013] In the preparation method described above, the mass ratio of the optical functional probe FTEBD to F127 in step 2 is 1:80 to 1:200.

[0014] In the preparation method described above, when R1 is a n-hexyl group and R2 is... The method for preparing the optical functional probe FTEBD includes the following steps: S1, 2-bromofluorene and bromo-substituted alkyl groups undergo a nucleophilic substitution reaction in the presence of tetrabutylammonium bromide to generate compound 2; S2, compound 2 and thiophene boric acid undergo Suzuki-Miyaura coupling under tetra(triphenylphosphine)palladium catalysis to generate compound 3; S3 and compound 3 undergo an NBS bromination reaction to generate compound 4; S4, compound 4, and tributyl(2,3-dihydrothiopheno[3,4-b][1,4]dioxane-5-yl)tinane undergo a Stille coupling reaction under tetra(triphenylphosphine)palladium catalysis to generate compound 5; S5, compound 5, reacts with n-butyllithium and tributyltin chloride to generate tin reagent, namely compound 6; S6, compound 6, and 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole undergo a Stille coupling reaction catalyzed by tetra(triphenylphosphine)palladium to generate compound 7; S7 and compound 7 undergo a reduction reaction in the presence of Fe powder and glacial acetic acid, reducing the nitro group to an amino group to obtain the target product probe FTEBD. The structural formulas of compounds 2-7 are as follows:

[0015] Formula II Formula III Formula IV

[0016] Formula V Formula VI

[0017] Formula VII In the preparation method described above, in step S1, 2-bromofluorene, bromohexane, and TBAB are placed in an alkaline solution and heated to react, and a dialkyl nucleophilic substitution reaction occurs at the 9-position of 2-bromofluorene.

[0018] In the preparation method described above, the bromine substitution position in step S3 during the bromination reaction is the α-position of the thiophene ring of compound 3.

[0019] In the preparation method described above, the molar ratio of compound 6 and 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole in step S6 is 2.1~2:1, the palladium catalyst is tetra(triphenylphosphine)palladium, the reaction temperature is 90-110 °C, and an inert gas N2 is used for protection.

[0020] As described above, the hydrated particle size microstructure of the F NPs is spherical, and the hydrodynamic particle size is concentrated in the range of 9 nm to 18 nm.

[0021] This application provides the application of the NO-activated organic nanosystem described above in dual-modal imaging of an inflammation model, which is used to prepare dual-modal imaging detection reagents for inflammation-related diseases, wherein the inflammation-related disease model is lipopolysaccharide (LPS)-induced acute inflammation.

[0022] As described above, the nanosystem F NPs specifically respond to NO. The excitation wavelength used for fluorescence spectroscopy acquisition and near-infrared II fluorescence imaging acquisition before and after NO activation is 808 nm, and the photoacoustic PA signal acquisition wavelength is 680 nm.

[0023] The organic nanosystem prepared in this application is coated with biocompatible biomolecules. Optical property studies show that before interaction with NO, the nanosystem exhibits weak ICT (Inductively Coupled Transmission) capabilities and no photoacoustic or near-infrared II (NIR-II) fluorescence signals due to the weak acceptor unit (benzothiadiazole diamine) in the activating group. Upon NO activation, the thiadiazole diamine group transforms into a strong acceptor benzotriazole derivative. The specific binding of the acceptor unit to NO in the molecular structure enhances the ICT effect, leading to a significant increase in photoacoustic and NIR-II fluorescence signals accompanied by a characteristic wavelength shift. NO, as a key regulatory molecule in inflammation, can reflect the occurrence, development, and resolution of inflammation through its dynamic changes. Therefore, utilizing the close relationship between NO levels and the monitoring of inflammatory diseases, real-time imaging of inflammatory sites can be achieved, providing an important tool for the diagnosis, mechanism research, and treatment evaluation of inflammation.

[0024] The nanosystem prepared by this invention has a uniformly distributed particle morphology with a size of about 15 nm (measured by dynamic light scattering), and the size is consistent with the size results obtained from the hydrated particle size.

[0025] The nanosystem prepared in this invention exhibits a distinct absorption peak in the 550 nm-900 nm wavelength range upon NO response, with a maximum absorption peak at 685 nm. The solution color changes from pale yellow to dark green after the response. Fluorescence spectroscopy results show that the nanosystem activates fluorescence in the NIR-II region upon NO response, demonstrating potential for near-infrared II fluorescence imaging. Photoacoustic signal analysis indicates that the 680 nm-840 nm photoacoustic signal is significantly activated upon NO response, indicating photoacoustic imaging capability. Selectivity experiments show that even in the presence of other analytes (1. NO2), the nanosystem exhibits good selectivity. - 2. NO3 - 3. ·OH, 4. H2O2, 5. ONOO - 6. O2 ·- 7. GSH, 8. ClO - 9. 1 The nanosystem (containing O2, 10. Blank, 11. NO, and 12. NO+ inhibitors) exhibits a specific response to NO, and its fluorescence emission is unaffected by other ions or other analytes, making it suitable for highly specific imaging of NO in vivo.

[0026] The response of the probe to NO described in this invention was also verified by theoretical calculations. The energy level parameters and energy level size of the highest occupied orbital (HOMO) and lowest unoccupied orbital (LUMO) of the molecule after the probe responds to NO were verified. This proved that the probe energy level decreased after the NO response, thus verifying the redshift of the absorption wavelength.

[0027] Therefore, the nanosystem of this invention enables photoacoustic and fluorescence-sensitive imaging of a mouse inflammation model. In a lipopolysaccharide (LPS)-induced acute inflammation model in mice, this nanosystem can achieve photoacoustic imaging of inflammation in the mouse leg and NIR-II fluorescence imaging of the mouse paw pads.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The organic nanosystem prepared by this invention is obtained by a biological macromolecule nanoprecipitation method. The preparation method is simple, has universality and good biocompatibility, and low toxicity and side effects.

[0029] 2. The nanosystem prepared by this invention can realize the activation of photoacoustic and NIR-II fluorescence signals under NO response, thereby achieving photoacoustic and NIR-II fluorescence imaging.

[0030] 3. The nanosystem prepared in this invention can achieve sensitive imaging of photoacoustic and NIR-II fluorescence in mouse muscles and footpads in an LPS-induced acute mouse inflammation model, and has obvious optical imaging signals compared with the phosphate-buffered saline (PBS) injection group. Attached Figure Description

[0031] Figure 1 This is the structural formula showing the change in molecular structure of the NO-activated probe before and after NO activation; Figure 2 This is a transmission electron microscope (TEM) image of the organic nanosystem F NPs prepared in Example 2. Figure 3 This is a dynamic light scattering (DLS) map of organic nanosystems F NPs. Figure 4 The image shows the UV absorption spectrum of the nanosystem F NPs in response to NO. The inset shows photographs of the solution before and after activation. Figure 5 The image shows the fluorescence emission spectrum of the nanosystem F NPs in response to NO. The inset shows the fluorescence imaging of the solution before and after activation. Figure 6 The image shows the photoacoustic spectrum of the nanosystem F NPs in response to NO. The inset shows the photoacoustic signal of the solution before and after activation. Figure 7 The image shows the fluorescence specificity selection signal of the nanosystem F NPs in response to NO. The inset shows the specific NIR-II fluorescence imaging of the material in response to NO in the presence of various analytes. Figure 8 This is a theoretical calculation before and after activating the FTEBD probe and the NO response; Figure 9 Photoacoustic imaging of nanosystems in a mouse acute inflammation model; Figure 10 This demonstrates the NIR-II fluorescence imaging capability of the nanosystem in an acute inflammation model of mouse paw pads. Figure 11 This is a schematic diagram of the nanosystem and NO-responsive dual-modal imaging of this application; Figure 12 This is the 1H NMR spectrum of the activation probe FTEBD in this application; Figure 13 This is the carbon NMR spectrum of the activation probe FTEBD in this application; Figure 14 This is the mass spectrometry of the activation probe FTEBD in this application. Detailed Implementation

[0032] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below. The specific embodiments listed below are merely descriptions of the principles and features of the present invention, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] All reagents used in the embodiments of this invention are commercially available, and the instruments used include: Dynamic light scattering: Zetasizer Nano ZS (Nano ZS90, Malvern); Transmission electron microscope: FEI Tecnai F20; UV-Vis spectrophotometer: UV-3600; Combined fluorescence spectrometer: Edinburgh FLS980; Photoacoustic imaging device: Multispectral Optoacoustic Tomography scanner (MSOT, iThera medical, Germany); Near-infrared two-window fluorescence imager: NIR-II imaging system (Suzhou Yingrui Optical Technology Co., LTD); The abbreviations for some chemical reagent names are: F127: Poloxamer is a polyoxyethylene polyoxypropylene ether block copolymer.

[0034] Example 1: Synthesis of the target probe FTEBD 1) Synthesis of Compound 2: Compound 1 (Comp. 1, 2-bromofluorene, 2 g, 8.16 mmol), tetrabutylammonium bromide (TBAB), bromohexane (5 g, 30 mmol), and 50% sodium hydroxide (NaOH, 15 mL) were mixed and reacted at 70 °C for 12 h. After the reaction was complete, the reaction solution was cooled to room temperature and extracted in dichloromethane (DCM) and saturated brine, and the organic layer was collected. Subsequently, the organic layer was dried over anhydrous sodium sulfate to remove the organic solvent, and the crude product was subjected to PE silica gel column chromatography to give compound 2 (3.34 g, yield: 99%), a pale yellow solid.

[0035] 2) Synthesis of Compound 3: Compound 2 (3.34 g, 8.08 mmol), thiophene boric acid (1.55 g, 12.12 mmol), tetrakis(triphenylphosphine)palladium (0.47 g, 0.4 mmol), and an aqueous solution of potassium carbonate (3.34 g, 24 mmol) were mixed in 1,4-dioxane and reacted at 95 °C for 12 h. After the reaction was completed, the reaction solution was cooled to room temperature and extracted with dichloromethane and saturated brine, and the organic layer was collected. Subsequently, the organic layer was dried over anhydrous sodium sulfate to remove the organic solvent. The crude product was subjected to PE / DCM (2:1) silica gel column chromatography to give compound 3 (2.69 g, yield: 80%) as a white solid.

[0036] 3) Synthesis of Compound 4: Compound 3 (2.69 g, 6.46 mmol) and NBS (2.30 g, 12.91 mmol) were mixed in THF and reacted at room temperature for 6 h. After the reaction was complete, the reaction solution was cooled to room temperature and extracted with dichloromethane and saturated brine, and the organic layer was collected. Subsequently, the organic layer was dried over anhydrous sodium sulfate to remove the organic solvent, and the crude product was subjected to PE / DCM (2:1) silica gel column chromatography to give compound 4 (3.20 mg, yield: 100%) as a white solid.

[0037] 4) Synthesis of Compound 5: Compound 4 (1.92 g, 3.88 mmol), tributyl(2,3-dihydrothiopheno[3,4-b][1,4]dioxane-5-yl)stanane (2 g, 4.64 mmol), and tetraphenylphosphine palladium (0.27 g, 0.23 mmol) were mixed in toluene and reacted at 95 °C for 12 h. After the reaction was completed, the reaction solution was cooled to room temperature and extracted with dichloromethane and saturated brine, and the organic layer was collected. Subsequently, the organic layer was dried over anhydrous sodium sulfate to remove the organic solvent, and the crude product was subjected to PE / DCM (1:1) silica gel column chromatography to give compound 5 (1.94 g, yield: 90%) as a yellow solid.

[0038] 5) Synthesis of Compound 6: Compound 5 (1.94 g, 3.48 mmol) was dissolved in THF solvent under N2 protection, and the mixture was then placed at -78 °C. When the mixture reached -78 °C, n-butyllithium (n-BuLi, 9 mL) was injected into the mixture using a syringe, and the reaction was continued at -78 °C for 2 h. Subsequently, tributyltin chloride (Bu3SnCl, 15 mL) was injected into the mixture using a syringe, and the mixture was reacted at room temperature for 12 h. After the reaction was complete, the organic solvent in the mixture was removed by vacuum rotary evaporation, followed by extraction with dichloromethane and saturated brine, and the organic layer was collected. After drying with anhydrous sodium sulfate, the organic solvent was removed by vacuum rotary evaporation to obtain the target compound 6 (2.95 g, yield: 100%).

[0039] 6) Synthesis of Compound 7: Compound 6 (2.95 g, 3.48 mmol) and 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole (0.65 g, 1.69 mmol) and tetrakis(triphenylphosphine)palladium (0.47 g, 0.4 mmol) were mixed in toluene and reacted at 100 °C for 24 h. After the reaction was complete, the reaction solution was cooled to room temperature and extracted with dichloromethane and saturated brine, and the organic layer was collected. Subsequently, the organic layer was dried over anhydrous sodium sulfate to remove the organic solvent, and the crude product was subjected to PE / DCM (1:2) silica gel column chromatography to give compound 7 (2.80 g, yield: 60%) as a dark blue solid.

[0040] 7) Synthesis of the target product probe F: Compound 7 (2.80 g, 2.09 mmol) and iron powder were mixed in glacial acetic acid (5 mL) and reacted at 100 °C for 8 h. After the reaction was completed, the reaction solution was cooled to room temperature and extracted with dichloromethane and saturated brine, and the organic layer was collected. Subsequently, the organic layer was dried over anhydrous sodium sulfate to remove the organic solvent, and the crude product was subjected to PE / DCM (1:3) silica gel column chromatography to obtain the yellow solid target product FTEBD (1.34 g, yield: 50%).

[0041] 1 H NMR (400 MHz, CDCl3): δ (ppm) 7.71-7.66 (m, 4H), 7.64-7.39 (m, 4H), 7.38-7.33 (m, 10H), 4.52-7.51 (d, J = 4Hz, 2H), 4.41-4.40 (d, J = 4Hz, 2H), 2.05-2.01 (t,J = 8Hz, 8H), 1.16-1.07 (m, 24H), 0.81-0.77 (t, J = 8Hz, 12H),0.73-0.61 (m, 8H). 13 C NMR (100 MHz, CDCl3): δ (ppm) 151.55, 150.98, 150.88,143.78, 140.67, 140.18, 139.21, 137.15, 133.34, 133.02, 127.07, 126.81,124.44, 124.31, 123.01, 122.87, 120.04, 119.73, 119.67, 114.22, 106.40,104.19, 65.08, 64.98, 55.18, 40.44, 31.49, 29.71, 23.72, 22.59, 14.01. MALDI-TOF MS (m / z) [M+H] + [C 76 H 83 N4O4S5] + Calculation result: 1275.501, found: 1275.554. The proton, carbon, and mass spectra of the target product FTEBD are shown below. Figure 12-14 As shown.

[0042] The synthesis route is shown below:

[0043] Example 2: Preparation of Nanoscale Systems The nanomaterial system was prepared using a nanoprecipitation method: Appropriate amounts of the probe and the biomacromolecule F127 (mass ratio 1:200) were weighed and dissolved completely in a suitable amount of THF solvent. Subsequently, under stirring, the mixture was injected into deionized water, repeatedly agitated several times, and then the THF in the solution was completely evaporated using N2. After ultrafiltration using a 30 kDa ultrafiltration tube, the final nanomaterial mother liquor (F NPs) was obtained.

[0044] The above organic nanosystem F NPs were subjected to transmission electron microscopy and dynamic light scattering, and the results are as follows: Figure 2 , 3 As shown. By Figure 2 It exhibits a spherical shape, composed of Figure 3 It can be seen that the nanosystem was successfully prepared, and the particle size of the prepared nanosystem is about 15 nm, with uniform distribution and size consistent with the size results obtained from the hydrated particle size.

[0045] Experimental Example 1: Detection of In Vitro Optical Signals Ultraviolet absorption spectroscopy detection: A 1 cm wide quartz cuvette was used, and 1 mL of 5 μM PBS solution containing nanomaterials was added. The cuvette containing the solution was placed in an ultraviolet absorption spectrometer for ultraviolet absorption spectroscopy acquisition. After the test, 1 μL of NO in PBS solution was added to the solution to bring the total NO concentration in the solution to 50 μM. The solution was then incubated in a 37 °C water bath for 30 min and placed in an ultraviolet absorption spectrometer again for ultraviolet absorption spectroscopy acquisition.

[0046] Figure 4 The image shows the UV absorption spectrum of the F NPs nanosystem after its response to NO, with the inset showing photographs of the solution before and after activation. As can be seen from the image, after the nanosystem responds to NO, a new characteristic absorption peak appears in the 550 nm-900 nm range, and the solution color changes from pale yellow to dark green.

[0047] Fluorescence spectroscopy detection: A 1 cm wide quartz fluorescein dish was used, and 1 mL of 5 μM PBS solution containing the nanomaterial was added. The dish containing the solution was placed in a fluorescence spectrometer for fluorescence emission spectroscopy acquisition. After the test, 1 μL of NO in PBS solution was added to the solution to bring the total NO concentration in the solution to 50 μM. The solution was then incubated in a 37 °C water bath for 30 min before being placed in the fluorescence spectrometer again for fluorescence emission spectroscopy acquisition.

[0048] Figure 5 This is the fluorescence emission spectrum of the nanosystem F NPs in response to NO, with the inset showing the solution fluorescence images before and after activation.

[0049] Photoacoustic spectroscopy and imaging acquisition: Both photoacoustic spectroscopy and image acquisition were performed using the MOST photoacoustic imaging system, with an excitation wavelength range of 680 nm-850 nm. The specific steps for in vitro photoacoustic data acquisition are as follows: A suitable amount of 5 μM PBS solution containing the nanomaterial was added to an in vitro photoacoustic test tube using a pipette. The ends of the test tube were then sealed, taking care to avoid air bubbles. The test tube containing the solution was placed in the photoacoustic imaging system for data acquisition. Data acquisition after a NO response involved adding a pre-incubated solution to the test tube.

[0050] Figure 6 The figure shows the photoacoustic spectrum of the F NPs nanosystem in response to NO, with the inset showing the photoacoustic signals of the solution before and after activation. As can be seen from the figure, the NIR-II fluorescence signal is activated after the nanosystem responds to NO, demonstrating sensitive photoacoustic and fluorescence imaging capabilities.

[0051] Specific fluorescence spectroscopy detection and imaging acquisition were selected: 12 sets of parallel solution samples (n = 3) were prepared to ensure that the concentration of nanomaterial F NPs was 5 μM, and different analytes (1. NO2) were used. - 2. NO3 - , 3. ·OH, 4. H2O2, 5. ONOO - 6.O2 ·- 7. GSH, 8. ClO - 9. 1 The concentration of O2, 10. Blank, 11. NO, and 12. NO+ inhibitors was 50 μM. The solution was incubated in a 37 °C water bath for 30 min and then placed in a fluorescence spectrometer and a near-infrared II small animal imager for fluorescence emission spectroscopy and imaging acquisition, respectively.

[0052] Figure 7 This is a fluorescence-specific selectivity signal diagram of the nanosystem F NPs in response to NO. The inset shows the specific NIR-II fluorescence imaging of the material's response to NO in the presence of various analytes. As can be seen from the figure, this nanosystem exhibits good selectivity after responding to NO, avoiding interference from other analytes and demonstrating potential for in vivo imaging.

[0053] Experiment Example 2: Animal Experiment All animal experiments were approved by the Animal Ethics Committee of Soochow University and conducted in accordance with the "Guidelines for the Care and Use of Laboratory Animals" (2024-N(A)-382). Eight-week-old female BALB / c mice were purchased from Gemma Pharmaceutical Technology Co., Ltd. For the lipopolysaccharide (LPS)-induced acute inflammation mouse model, mice were subcutaneously injected with LPS (5 mg / mL, 50 μL) and maintained for 8 hours. Swelling and redness at the injection site indicated successful model establishment. The PBS solution (200 μM, 50 μL) used for the nanomaterials was the ultrafiltration stock solution diluted to the appropriate concentration with PBS before use.

[0054] In vivo photoacoustic imaging data acquisition: LPS solution (5 mg / mL, 50 μL) was injected into the left and right thigh muscles of mice, respectively. Eight h later, PBS solution (50 μL) was injected into the left thigh muscle, and PBS solution of nanomaterials (200 μM, 50 μL) was injected into the right thigh muscle. Thirty min later, the mice were placed in a photoacoustic imaging system under anesthesia for data acquisition, and photoacoustic data at 680 nm excitation were collected.

[0055] In vivo fluorescence imaging data acquisition: LPS solution (5 mg / mL, 50 μL) was injected into the left and right paw pads of mice, respectively. Eight h later, PBS solution (20 μL) was injected into the left paw pad, and PBS solution of nanomaterials (200 μM, 20 μL) was injected into the right paw pad. Thirty min later, the mice were placed in a near-infrared II small animal imaging system under anesthesia for data acquisition.

[0056] Figure 9 This is photoacoustic imaging of the nanosystem in a mouse acute inflammation model. As shown in the figure, the nanosystem can achieve photoacoustic imaging of inflammation in the thigh muscles of mice, with obvious photoacoustic signals at the inflamed site, while the control group (PBS group) has almost no signal.

[0057] Figure 10 This demonstrates the NIR-II fluorescence imaging capability of the nanosystem in an acute inflammation model of mouse paw pads. As shown in the figure, the nanosystem can achieve near-infrared II fluorescence imaging of the mouse paw pads, while the control group (PBS group) shows almost no fluorescence signal, indicating that the nanosystem can achieve dual-modal imaging capability of inflammation at the in vivo level in mice.

[0058] This invention discloses a novel NO-activated near-infrared II organic semiconductor probe, whose main component is a benzothiadiazole diamine derivative. It further incorporates an electron-donating group fluorene and conjugated extension fragments thiophene and dioxanethiophene to form a probe with a D-π-A-π-D structure. In the inactive state, due to the suppression of ICT (Inductively Coupled Transmission), its photoacoustic (PA) and near-infrared II (NIR-II, 900–1200 nm) fluorescence signals are turned off. Upon response to NO, the weak electron acceptor (benzo[c][1,2,5]thiadiazole-5,6-diamine) is specifically oxidized to a strong electron acceptor (5H-[1,2,3]triazole[4,5-f]-2,1,3-benzothiadiazole), enhancing intramolecular electron transfer and leading to an extended absorption wavelength. This activates the photoacoustic and near-infrared II (NIR-II) fluorescence signals, achieving specific activation of photoacoustic and NIR-II fluorescence. To improve the biocompatibility of the probe, a nanosystem with good water solubility and biocompatibility was prepared by nanoprecipitation. This nanosystem can achieve a sensitive response to NO and realize high-sensitivity detection.

[0059] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A nitric oxide-activated organic nanosystem, characterized in that: comprising a photo-functional probe FTEBD and an amphiphilic block copolymer PEG- b -PPG- b -PEG The nano-system is prepared by nano-precipitation method, the activated organic nano-system can specifically recognize NO in the microenvironment of the inflammation site and generate a detectable optical signal, and the structural formula of the photo-functional probe FTEBD is: Formula I Where R1 is or R2 is or or .

2. The method for preparing a nitric oxide-activated organic nanosystem according to claim 1, characterized in that... Includes the following steps: Step 1: Fabrication of the optical functional probe FTEBD; Step 2: Weigh the probe FTEBD and the amphiphilic block copolymer F127 PEG- b -PPG- b -PEG was used to prepare NO-activated organic nano-systems F NPs via nanoprecipitation. The specific steps are as follows: Organic solvent was added to probes FTEBD and F127 to dissolve them, and the mixture was stirred to form a homogeneous solution. The solution was then rapidly injected into deionized water, repeatedly agitated, and sonicated to ensure uniform dispersion of the nanoparticles. Subsequently, the liquid surface was agitated with airflow to completely remove the organic solvent. The solution was then concentrated by centrifugation using an ultrafiltration tube to obtain an aqueous dispersion mother liquor of F NPs.

3. The method for preparing a nitric oxide-activated organic nanosystem according to claim 2, characterized in that: In step 2, the mass ratio of the optical functional probe FTEBD to F127 is 1:80~200.

4. The method for preparing a nitric oxide-activated organic nanosystem according to claim 2, characterized in that: When R1 is a n-hexyl group, R2 is... The method for preparing the optical functional probe FTEBD includes the following steps: S1, 2-bromofluorene and bromo-substituted alkyl groups undergo a nucleophilic substitution reaction in the presence of tetrabutylammonium bromide to generate compound 2; S2, compound 2 and thiophene boric acid undergo Suzuki-Miyaura coupling under tetra(triphenylphosphine)palladium catalysis to generate compound 3; S3 and compound 3 undergo an NBS bromination reaction to generate compound 4; S4, compound 4, and tributyl(2,3-dihydrothiopheno[3,4-b][1,4]dioxane-5-yl)tinane undergo a Stille coupling reaction under tetra(triphenylphosphine)palladium catalysis to generate compound 5; S5, compound 5, reacts with n-butyllithium and tributyltin chloride to generate tin reagent, namely compound 6; S6, compound 6, and 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole undergo a Stille coupling reaction catalyzed by tetra(triphenylphosphine)palladium to generate compound 7; S7 and compound 7 undergo a reduction reaction in the presence of Fe powder and glacial acetic acid, reducing the nitro group to an amino group to obtain the target product probe FTEBD. The structural formulas of compounds 2-7 are as follows: Formula II Formula III Formula IV Formula V Formula VI Formula VII.

5. The method for preparing a nitric oxide-activated organic nanosystem according to claim 4, characterized in that: In step S1, 2-bromofluorene, bromohexane, and TBAB are heated in an alkaline solution to react, and a dialkyl nucleophilic substitution reaction occurs at the 9-position of 2-bromofluorene.

6. The method for preparing a nitric oxide-activated organic nanosystem according to claim 4, characterized in that: In step S3, the bromine in the bromination reaction is substituted at the α-position of the thiophene ring of compound 3.

7. The method for preparing a nitric oxide-activated organic nanosystem according to claim 4, characterized in that: In step S6, the molar ratio of compound 6 and 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole is 2.1~2:

1. The palladium catalyst is tetra(triphenylphosphine)palladium, the reaction temperature is 90-110 °C, and an inert gas is used for protection.

8. The method for preparing a nitric oxide-activated organic nanosystem according to claim 1, characterized in that: The organic nanosystem F NPs exhibits a spherical hydration particle size microstructure, with hydrodynamic particle sizes concentrated between 9 nm and 18 nm.

9. The application of the nitric oxide-activated organic nanosystem according to claim 1, characterized in that: This is used to prepare a dual-modal imaging detection reagent for inflammation-related diseases, wherein the inflammation-related diseases are lipopolysaccharide (LPS)-induced acute inflammation.

10. The application of the nitric oxide-activated organic nanosystem according to claim 9, characterized in that: The nanosystem F NPs specifically respond to NO. The excitation wavelength used for fluorescence spectroscopy acquisition and near-infrared II fluorescence imaging acquisition before and after NO activation is 808 nm, and the photoacoustic PA signal acquisition wavelength is 680 nm.

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