An organic nanomaterial, a preparation method and application thereof

Abstract

The present application relates to an organic nanomaterial, a preparation method and an application, and the organic nanomaterial is 6TCl-PS NPs, PTQ-PS NPs, Y8-PS NPs or TQNH-PS NPs. The organic nanomaterial has high fluorescence quantum yield and good biocompatibility; can realize long-term in-vivo circulation, and can realize high-efficiency enrichment at a tumor site through an enhanced permeability and retention (EPR) effect, and can realize NIR-II high-signal-to-noise ratio and high-resolution fluorescence imaging of a mouse blood vessel, a lymph and a tumor under conditions of low-power laser (1 W / cm 2 ) and short exposure time (5 ms).

Description

Technical Field

[0001] This invention relates to the field of organic nanomaterials technology, and more particularly to an organic nanomaterial, its preparation method, and its application. Background Technology

[0002] For near-infrared II (NIR-II) light-emitting organic molecules, enhanced intramolecular charge transfer processes can boost long-wavelength absorption but also increase interactions with other molecules in the surrounding environment, especially water molecules, thus weakening fluorescence emission. Therefore, constructing organic fluorophores with NIR-II emission without sacrificing brightness is a key challenge in developing NIR-II organic fluorescent materials with clinical potential.

[0003] The design strategies for high-brightness NIR-II organic fluorescent materials that have been reported and validated include: (1) constructing steric hindrance; (2) introducing shielding units; (3) suppressing TICT (twisted intramolecular charge transfer) processes; (4) utilizing protein encapsulation; and (5) constructing AIE structure molecules. Numerous studies have effectively enhanced the in vivo imaging brightness of NIR-II organic fluorescent materials based on these strategies, providing inspiration and food for thought for further research in this field. Clearly, strategies that enhance fluorescence brightness through molecular structure modification and tailoring inevitably have far-reaching consequences for NIR-II molecules, causing changes in photophysical properties, making the optimization process complex and specific. Similarly, strategies that enhance brightness by screening guest molecules to form supramolecular assembly systems also suffer from limitations, requiring specific host-guest pairs to pair successfully, lacking practical applicability. Therefore, exploring convenient, efficient, and universally applicable strategies for amplifying the fluorescence brightness of NIR-II organic nanoprobes in aqueous media is of great significance.

[0004] In 2018, Dai Hongjie's team at Stanford University designed an organic NIR-II dye, FE. Considering that FE exhibits high QY (fluorescence quantum yield) in the organic solvent toluene, Dai et al. encapsulated FE within the hydrophobic interior of the amphiphilic polymer poly(styrene-chloromethylstyrene)-grafted-polyethylene glycol (PS-g-PEG). The PS backbone forms a hydrophobic core, mimicking the toluene environment to encapsulate the dye and prevent its aggregation and fluorescence quenching. The hydrophilic PEG chain endows p-FE with water solubility and biocompatibility, ultimately obtaining nanofluorophores with a QY as high as 16.5% in an aqueous environment. They successfully used p-FE to achieve non-invasive real-time blood flow tracking in the mouse brain and in vivo dual-color imaging of tumors. Similarly, in 2022, Xiong Hu's research group at Nankai University developed two BODIPY-based DAD-type fluorescent probes, NK1133 and NK1143. NK1143 was obtained by grafting a 4-tert-butylphenyl group onto the receptor core of NK1133 to inhibit H aggregation. Notably, the team also synthesized an additional steric hindrance, SC12, which possesses a benzene ring and a long alkyl chain at the tail. The co-assembled NK1143-SC1NPs, under the combined regulation of the shielding group 4-tert-butylphenyl group and the steric hindrance SC12, exhibited fluorescence brightness 353 times higher than NK113. Under 980 nm excitation, they achieved 8 mm deep light penetration and high-resolution NIR-II fluorescence imaging of blood vessels, with a signal-to-noise ratio as high as 7.8 / 1. In 2024, Fan Quli's team at Nanjing University of Posts and Telecommunications proposed a strategy to enhance fluorescence by creating an alkyl atmosphere within perylene diimide derivative-based nanoparticles (OPE-PDI). Verification through structural characterization and theoretical calculations showed that OPE-PDI exhibited stronger fluorescence in n-hexane (HE) than in other solvents. Inspired by this, the team co-encapsulated OPE-PDI with octadecane (a long alkyl chain), a homologue of HE, and DSPE-mPEG to enhance its fluorescence brightness by creating an alkyl atmosphere in the nanoparticles, and further evaluated its imaging performance.

[0005] The aforementioned technologies share a common core: leveraging the excellent fluorescence properties of small-molecule dyes in a specific solvent, they utilize steric hindrance agents to simulate this solvent atmosphere around the nanoparticle core, ultimately achieving an effective enhancement of the brightness of near-infrared organic probes. However, these technologies fail to simultaneously elucidate the brightness enhancement mechanism and expand the application of nanomaterials in the near-infrared II region, and therefore require further improvement.

[0006] It should be noted that this section is intended to provide background or context for the technical solutions of the invention as set forth in the claims. The description herein does not imply acceptance as prior art simply because it is included in this section. Summary of the Invention

[0007] The purpose of this invention is to provide an organic nanomaterial, a preparation method, and an application, thereby at least partially solving one or more problems caused by the limitations and defects of related technologies.

[0008] The present invention first provides an organic nanomaterial, wherein the organic nanomaterial is: 6TCl-PS NPs, PTTQ-PS NPs, Y8-PS NPs or TQNH-PS NPs.

[0009] The present invention further provides a method for preparing organic nanomaterials, the method comprising: S1, Dissolve the solid organic molecule in tetrahydrofuran to obtain a molecular stock solution, wherein the organic molecule is 6TCl, PF-TTQ, Y8 or TTQ-NH; S2, PS 2000 -PEG 2000 The solid was dissolved in tetrahydrofuran to obtain a coating agent stock solution; S3. Mix the molecular stock solution and the coating agent stock solution evenly, then add pure water to remove tetrahydrofuran from the solution. After filtration, a nanoparticle solution is obtained, which is an organic nanomaterial.

[0010] In this invention, in S1, the concentration of the molecular stock solution is 1 mg / mL.

[0011] In this invention, in step S2, the concentration of the coating agent stock solution is 5 mg / mL.

[0012] In this invention, in S3, the volume ratio of the mixture consisting of the molecular stock solution and the coating agent stock solution to pure water is 1:9.

[0013] In this invention, in step S3, the solution is filtered three times using a 0.22 μm filter membrane.

[0014] The present invention also provides an application of an organic nanomaterial, which is used for the fabrication of nanoprobes.

[0015] In this invention, the nanoprobe is used for in vivo imaging.

[0016] The technical solution provided by this invention may include the following beneficial effects: The organic nanomaterials of this invention exhibit high fluorescence quantum yield and good biocompatibility; they can achieve long-term in vivo circulation and can be efficiently enriched at tumor sites through enhanced permeation and retention (EPR) effects, even under low-power laser (1 W / cm²) conditions. 2 With a short exposure time (5 ms), high signal-to-noise ratio and high resolution fluorescence imaging of mouse blood vessels, lymph nodes and tumors can be achieved using NIR-II. Attached Figure Description

[0017] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this disclosure and, together with the description, serve to explain the principles of this disclosure. It is obvious that the drawings described below are merely some embodiments of this disclosure, and those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0018] Figure 1 This illustrates the analytical content of the present invention; Figure 2 The synthesis equation of the 6TCl of the present invention is shown; Figure 3 The 1H NMR spectrum of compound 6TCl in this invention is shown; Figure 4 The high-resolution mass spectrum of compound 6TCl in this invention is shown. Figure 5 (a) RB3LYP 6-31 ground-state geometry of optimized 6TCl at the G(D,P) level; (b) calculated electrostatic potential surface of the optimized 6TCl molecule; (c) HOMO and LUMO energy levels and electron distribution of optimized 6TCl. Figure 6 This diagram illustrates the optimal solvent selection process for 6TCl in this invention. Figure 7 (a) Absorption spectra of 6TCl molecules in different solvents. The absorbance at 808 nm is 0.41 in all solvents; (b) Fluorescence spectra of each solution in a; (c) Fluorescence imaging and quantification of 1 mL of the three solutions. Dark-field fluorescence images: (λex: 808 nm, 1000 nm LP, 5 ms); Figure 8 (a) Femtosecond transient absorption spectra of 6TCl in different solvents (top) and curves (bottom). 6TCl was excited using an 808 nm femtosecond pulsed laser. Arrows indicate the trend of the transient absorption spectrum over time; (b) Representative kinetic curves within the GSB region; (c) Global fitting results of the multi-exponential model; Figure 9 A schematic diagram of the preparation of 6TCl-DSPE NPs and 6TCl-PS NPs nanoparticles is shown. Figure 10 (a) Particle size distribution (DLS) of 6TCl-PS NPs. Inset: Solution image of 6TCl-PS NPs; (b) Particle size distribution of 6TCl-DSPE NPs by DLS and surface morphology by TEM; Inset: Solution image of 6TCl-DSPE NPs; (c) Bright-field and dark-field images of five solutions: ① pure water; ② DSPE-PEG2000 aqueous solution (1 mL, 10 μg / mL); ③ PS2000-PEG2000 aqueous solution (1 mL, 10 μg / mL); ④ 6TCl-DSPE NPs solution (1 mL, OD = 0.73); ⑤ 6TCl-PS NP solution (1 mL, OD = 0.73). Top: Bright-field image; Bottom: Dark-field image under 808 nm excitation (λex: 808 nm, 1000 nm LP, 20 ms). (d) Absorption spectra of the two nanoparticles (OD808 nm = 0.73); (e) Emission spectra of two nanoparticle solutions under 808 nm excitation; (f) Quantitative analysis results of fluorescence intensity of the five solutions in c; Figure 11 The graph shows the quantum yield measurement results of 6TCl-DSPE NPs and 6TCl-PS NPs in this invention; Figure 12 (a) Schematic diagram of the composition ratio of organic fluorescent molecules and amphiphilic polymer coating agents in 6TCl-PS NPs; (b) Different m 6TCl :m PS2000-PEG2000 Absorption spectrum (OD) of nanoparticle solution 808 nm = 0.79); (c) Emission spectrum of solution b (λ) ex = 808 nm); (d) Bright-field and dark-field images of 1 mL solutions in b. Top: Bright-field image. Bottom: Dark-field image with 808 nm excitation (λ). ex : 808 nm, 1000 nmLP, 4 ms); Quantitative analysis of fluorescence intensity in each solution of (e)d, at different m 6TCl :m PS2000-PEG2000 Absorption spectrum of nanoparticle solution ( 7 μg / mL); (f) Different m 6TCl :m PS2000-PEG2000 Absorption spectrum of nanoparticle solution ( 7 μg / mL); Emission spectrum of the solution in (g)f (λex = 808 nm); Bright-field and dark-field images of 1 mL solutions in (h)f. Top: Bright-field image. Bottom: Dark-field image with 808 nm excitation (λ). ex : 808 nm, 1000 nmLP, 4 ms); (i) Quantitative analysis results of fluorescence intensity of each solution in h; Figure 13 The chemical structures of the organic molecules PF-TTQ, Y8, and TTQ-NH are shown. Figure 14 The NIR-II fluorescence quantum yields of benzene ring-doped and non-benzene ring-doped nanoparticles are shown. Figure 15 Quantum yield measurement of PTTQ-DSPE NPs and PTTQ-PS NPs: concentration OD set for each solution 808 nm =0.02, 0.04, 0.06, 0.08, 0.10; Fluorescence spectrum integration range: ( 1000 nm, 1700 nm): (a) Absorption spectrum of PTTQ-DSPE NPs in water; (b) Corresponding fluorescence spectrum of PTTQ-DSPE NPs; (c) Curve of integral fluorescence intensity of PTTQ-DSPE NPs in water under 808 nm excitation as a function of absorbance; (d) Absorption spectrum of PTTQ-PS NPs in water; (e) Corresponding fluorescence spectrum of PTTQ-PS NPs; (f) Curve of integral fluorescence intensity of PTTQ-PS NPs in water under 808 nm excitation as a function of absorbance. Figure 16 Quantum yield measurements of Y8-DSPE NPs and Y8-PS NPs are shown. Concentration OD is set for each solution. 808 nm =0.02, 0.04, 0.06, 0.08, 0.10. Fluorescence spectrum integration range: ( 1000 nm, (1700 nm). (a) Absorption spectrum of Y8-DSPE NPs in water; (b) Corresponding fluorescence spectrum of Y8-DSPE NPs; (c) Curve of integrated fluorescence intensity of Y8-DSPE NPs in water under 808 nm excitation as a function of absorbance; (d) Absorption spectrum of Y8-PS NPs in water; (e) Corresponding fluorescence spectrum of Y8-PS NPs; (f) Curve of integrated fluorescence intensity of Y8-PS NPs in water under 808 nm excitation as a function of absorbance; Figure 17 Quantum yield measurements of TQNH-DSPE NPs and TQNH-PS NPs are shown. Concentration OD is set for each solution. 808 nm = 0.02, 0.04, 0.06, 0.08, 0.10. Fluorescence spectrum integration range: ( 1000 nm, (1700 nm). (a) Absorption spectrum of TQNH-DSPE NPs in water. (b) Corresponding fluorescence spectrum of TQNH-DSPE NPs. (c) Curve of integrated fluorescence intensity of TQNH-DSPE NPs in water under 808 nm excitation as a function of absorbance. (d) Absorption spectrum of TQNH-PS NPs in water. (e) Corresponding fluorescence spectrum of TQNH-PS NPs. (f) Curve of integrated fluorescence intensity of TQNH-PS NPs in water under 808 nm excitation as a function of absorbance; Figure 18 Showing 6TCl-PS 200 Surface morphology and particle size distribution of NPs; Figure 19 Showing 6TCl-PS 200 Determination of the fluorescence quantum yield of NPs. Setting the solution concentration OD... 808 nm = 0.02, 0.04, 0.06, 0.08, 0.10. Fluorescence spectrum integration range: ( 1000 nm, 1700 nm). (a) 6TCl-PS 200 (a) Absorption spectrum of NPs in water; (b) 6TCl-PS 200 (c) Corresponding fluorescence spectra of NPs; 6TCl-PS in water 200 Curves showing the integral fluorescence intensity of NPs under 808 nm excitation as a function of absorbance; Figure 20 Showing 6TCl-PS 200 Results of the effect of NPs on cell viability of MC3T3 cells; Figure 21 The image shown is (a) a mouse NIR-II vascular image (λ). ex 808 nm, 1 W / cm 2 (a) 1000 nm LP, 5 ms); (b) Fluorescence intensity fitting curve of blood vessel cross section in Figure a. (c) Schematic diagram of injection site for mouse lymph node imaging; (d) Fluorescence intensity fitting curve of lymph node cross section in Figure e; (e) NIR-II lymph node image of mouse. Left hind limb: 20 μL, 250 μg / mL ICG in PBS solution. Right hind limb: 20 μL, 250 μg / mL 6TCl-PS 200 NPs in PBS solution (λ) ex 808 nm, 1 W / cm 2 (1000 nm LP, 5 ms); Figure 22 (a) 6TCl-PS at different time points 200NPs-mediated NIR-II tumor imaging in mice. (λex: 808 nm, 1 W / cm²) 2 (a) Fluorescence intensity changes over time at the tumor site; (b) NIR-II fluorescence imaging images of isolated organs; (c) Quantitative analysis of fluorescence signals in isolated organs; Figure 23 The results of H&E staining of the major organs of the mouse are shown. Detailed Implementation

[0019] Exemplary embodiments will now be described more fully with reference to the accompanying drawings. However, these exemplary embodiments can be implemented in many forms and should not be construed as limited to the examples set forth herein; rather, they are provided so that the invention will be more comprehensive and complete, and will fully convey the concept of the exemplary embodiments to those skilled in the art. The described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0020] Furthermore, the accompanying drawings are merely illustrative diagrams of embodiments of the present invention and are not necessarily drawn to scale. The same reference numerals in the drawings denote the same or similar parts, and therefore repeated descriptions of them will be omitted. Some block diagrams shown in the drawings are functional entities and do not necessarily correspond to physically or logically independent entities.

[0021] This invention proposes a simple, efficient, and universal benzene ring doping strategy to enhance fluorescence by simulating a toluene solvent atmosphere within conjugated small molecule-based nanoparticles, thereby improving NIR-II fluorescence imaging. First, the fluorescence performance of the small molecule 6TCl in different solvents was investigated, revealing that it exhibits the highest fluorescence intensity in toluene. Subsequently, excited-state kinetic analysis using ultrafast spectroscopy revealed the intrinsic mechanism of fluorescence enhancement in toluene: the hydrophobic and planar rigid benzene ring in toluene can regulate the molecular stacking mode and aggregation environment within the nanostructure, thereby reducing fluorescence quenching caused by aggregation and prolonging the lifetime of radiative transition components. Therefore, this application selects the amphiphilic polymer PS, whose molecular backbone contains benzene ring motifs. 2000 -PEG 2000 A benzene ring-doped nanoparticle strategy was proposed to simulate a toluene solvent environment for constructing benzene ring-doped nanoparticles. 6TCl-PS NPs exhibited a 4.3-fold increase in fluorescence intensity compared to undoped 6TCl-DSPE NPs, fully demonstrating the effectiveness of the strategy. Optimizing the mass ratio of the coating agent to organic molecules in the nanostructure yielded benzene ring-doped nanoparticles, 6TCl-PS, with a fluorescence quantum yield as high as 40%. 200NPs. Furthermore, three additional molecules (PF-TTQ, Y8, and TTQ-NH) were selected as representative organic molecules to construct benzene ring-doped nanoparticle models. Compared with the control group, their fluorescence quantum yields were enhanced by 2.32, 1.10, and 1.83 times, respectively, strongly demonstrating the universality of this strategy for different structural molecules. Finally, 6TCl-PS... 200 NPs assist in achieving high-resolution, high-contrast in vivo vascular, lymphatic, and tumor imaging in NIR-II.

[0022] The specific implementation process is as follows: 1. Experimental reagents Table 1 Main experimental materials and reagents

[0023] 2. Experimental apparatus Table 2 Experimental Instruments

[0024] 3. Synthesis of the organic molecule 6TCl Synthesis of π-conjugated oligomers (6TCl) via Knoevenagel condensation reaction can be found in [reference needed]. NANOSCALE HORIZONS 2021, 6 (2), 177-185. records.

[0025] Specifically, a certain amount of 6,6,12,1-tetra(4-hexylphenyl)-1,dihydro-thieno[2'',3'':4',5']thieno[3',2':4,5]cyclopentadieno[1,b]thieno[2',3':4,5]cyclopentadieno[1'',2'':4,5]thieno-2,8-dicarboxaldehyde and a certain amount of 2-(5,6-dichloro-3-oxo-2,3-dihydro-1H-indene-1-ylidene)malonitrile were placed in a 500 mL double-necked round-bottom flask. After purging with nitrogen three times, anhydrous chloroform (250 mL) was added under an inert atmosphere to dissolve the flask. Then, pyridine (6.25 mL) was slowly added dropwise as a catalyst using a syringe. A condenser was installed, and the reaction was carried out in an oil bath with constant temperature and magnetic stirring for 24 hours. After the reaction was completed, the system was cooled to room temperature, and the solvent was removed by rotary evaporation. The residue was then precipitated by a methanol gradient (100 mL) and purified by silica gel column chromatography to finally obtain the target product 6TCl, which was a blue solid (2 g, 86%).

[0026] 4. Molecular calculation methods (1) Theoretical basis All quantum chemical calculations in this invention are based on density functional theory (DFT). DFT is a quantum mechanical method that uses electron density as the fundamental variable to study the ground-state properties of multi-electron systems. Its core advantage lies in avoiding the complex multi-electron wavefunction calculations in traditional wavefunction theory, significantly improving efficiency while maintaining computational accuracy, and is particularly suitable for the medium-sized organic molecular systems in this application.

[0027] The calculations employed the B3LYP hybrid functional, which incorporates the Hartree-Fock exchange energy and the DFT correlation energy. This functional is one of the most classic and widely used functionals for studying the electronic structure, geometry, and energy of organic molecules. It strikes a good balance between computational cost and accuracy, demonstrating excellent reliability in predicting molecular geometry, vibrational frequencies, and leading-edge orbital energies.

[0028] (2) Basis set and computational level All calculations were performed at the RB3LYP / 6-31G(D,P) theoretical level. Here, RB3LYP represents the use of the B3LYP functional within a restricted framework, applicable to calculations of closed-shell molecules (such as the ground state S0). 6-31G(D,P) is the standard split-valence basis set, adding d-polarization functions for atoms other than hydrogen and p-polarization functions for hydrogen. The introduction of polarization functions is crucial for accurately describing the polarization of chemical bonds and the shape of molecular orbitals, and is a commonly used basis set for optimizing geometry and calculating electronic properties.

[0029] All calculations were performed using the Gaussian 16 quantum chemistry software package. Data post-processing and visualization were performed using GaussView 6.0.

[0030] 5. Optical performance testing (1) Test conditions Considering the absorption peak of the material and the operating wavelength of mainstream lasers, an 808 nm laser was selected as the excitation wavelength for this experiment to determine the optical properties of the material. Furthermore, the 808 nm laser (1 W / cm²)... 2 This material can function well within biological tissues, ensuring high imaging quality without harming mice. To accurately evaluate the intrinsic fluorescence properties of the material in different solution systems, this study employed the isoabsorbance method for comparative analysis based on the Lamber-Beer law (Equation 1). According to the Lamber-Beer law, absorbance A is defined as: (Equation 1) in, The molar extinction coefficient is . For solution concentration, optical path length In absorption spectroscopy, the thickness of the cuvette is typically used. Therefore, absorbance A directly reflects the total amount of photons absorbed by the sample at a specific wavelength.

[0031] fluorescence intensity The number of absorbed photons is related to the quantum yield. The basic formula (Equation 2) is: (Equation 2) in, To excite the intensity of light, The light absorption rate is the proportion of light absorbed by the sample. Let A be the fluorescence quantum yield, and K be the instrument constant. In the experiment, the absorbance A was controlled within a low range. According to the Taylor series expansion, when... When I was very young, Simplifying and rearranging equations 1 and 2, we obtain equation 3: (Equation 3) According to Equation 3, fluorescence intensity Absorbance A and fluorescence quantum yield The following relationship applies between them: (Equation 4) In the experiment, under the same test conditions, this application tested the sample at the excitation wavelength ( The absorbance at 8 nm was adjusted to the same value. At this point, the number of excitation photons absorbed by different samples remained consistent. Based on this premise, the difference in the integral area or peak intensity of the measured fluorescence spectrum directly reflects the difference in quantum yield of the material in different solutions. This method effectively eliminates the concentration effect, ensuring the reliability and scientific rigor of fluorescence performance comparisons.

[0032] (2) Determination of fluorescence quantum yield The fluorescence quantum yield is the ratio of photons absorbed by a substance to photons emitted through fluorescence. Its value is typically less than 1. F The higher the value of Φ, the stronger the fluorescence of the compound, while the fluorescence quantum yield of non-fluorescent substances is equal to or very close to zero. Accurate determination of Φ... FCharacterizing the fluorescence properties of materials is essential. Methods for determining fluorescence quantum yield are divided into absolute and relative methods. Absolute quantum yield measurements are limited by instrument configuration, making it difficult to test long-wavelength fluorescent emitting materials, and are also expensive. Relative quantum yield measurements do not require complex and expensive instruments and are one of the most widely used methods. Therefore, this study uses the relative method to test the fluorescence quantum yield of materials. The relative method uses a standard fluorescent material with a known fluorescence quantum yield as a reference. Under the same experimental conditions, the absorption and fluorescence emission spectra of the standard sample and the test sample are measured separately. The fluorescence quantum yield of the test sample is obtained by converting the ratio of the integrated fluorescence intensity to the absorbance of the two samples.

[0033] Under dilute solution conditions, the number of photons absorbed by the sample at the excitation wavelength is proportional to its absorbance A; the number of photons emitted is proportional to the integrated area I of its fluorescence emission spectrum (fluorescence intensity integrated over the emission wavelength range). Therefore, the fluorescence quantum yields of the test sample (X) and the standard sample (ST) satisfy: (Equation 5) Among them, Ф X With Ф ST The fluorescence quantum yields of the test sample and the standard sample are respectively; k X With k ST A represents the slope of the linear fit of the integrated fluorescence intensity of the fluorescence emission spectrum measured under the same excitation wavelength and instrument parameters. X With A ST η represents the absorbance of both at the excitation wavelength. X With η ST Here, represents the refractive index of the solvent in the test system and the standard system, respectively. When the absorbance of the test sample and the standard sample are the same, the above formula can be simplified to: (Equation 6) To minimize the impact of internal filtration and self-absorption effects on the measurement results, a 10 mm optical path cuvette was used during the test. The sample solution was diluted during the test to ensure its absorbance at the excitation wavelength. .

[0034] Based on the above testing principle, IR-1061 (λ) with a fluorescence spectrum that overlaps to some extent with the sample to be tested was selected. abs = 1061nm, λ em = 1100 nm, Φ in dichloromethane 1R-1061Using a standard sample (0.59%), the standard solution and the test solution were diluted with the corresponding solvents to obtain absorbance (OD) values ​​of 0.02, 0.04, 0.06, 0.08, and 0.10 at 808 nm. Fluorescence spectra of the solutions with OD values ​​from 0.02 to 0.10 were measured under 808 nm excitation. The integrated fluorescence spectral area was calculated within the integrated domain of 1000 nm to 1700 nm, and a linear fitting curve of the integrated value versus absorbance was plotted and the slope was recorded. Finally, the relative fluorescence quantum yield of the test material was obtained by substituting the values ​​into Equation 6.

[0035] 6. Preparation of Nanoparticles This experiment used a nanoprecipitation method to prepare 6TCl-DSPE NPs and 6TCl-PS NPs. 1 mg of 6TCl solid and 10 mg of DSPE-PEG were accurately weighed using an electronic balance. 2000 Powder with 10 mg PS 2000 -PEG 2000 Solids were prepared and dissolved separately in tetrahydrofuran to obtain a molecular stock solution (1 mg / mL, 1 mL) and two coating agent stock solutions (5 mg / mL, 2 mL). 400 μL of the molecular stock solution and 1.6 mL of the coating agent stock solution were mixed, ensuring a molecular to coating agent mass ratio of 1:20, and ultrasonically mixed. For the nanoprecipitation method, the mixture and pure water must have a volume ratio of 1:9. 18 mL of pure water was taken, and 2 mL of the mixture was rapidly injected into the pure water under ultrasonic conditions. Ultrasonication was continued for 35 minutes, with the entire process performed in an ice bath. After ultrasonication, the mixture was allowed to stand overnight to allow the remaining tetrahydrofuran to completely evaporate. The next day, the solution was filtered three times through a 0.22 μm filter membrane, and the clear, transparent nanoparticle filtrate containing 6TCl-DSPE NPs and 6TCl-PS NPs was collected. The solution was then concentrated in an ultracentrifuge at 5000 rpm for 10 min, and the concentrate was stored at 4°C for later use.

[0036] 7. Cytotoxicity test In this experiment, the MTT assay was used to determine cytotoxicity. Mouse embryonic osteoblasts (MC3T3) were cultured in α-MEM medium (10% fetal bovine serum, 100 μg / mL penicillin, and 0.1 mg / mL streptomycin) at 37°C in a 5% CO2 incubator. First, 100 μL of cell suspension was seeded into 96-well plates and pre-incubated in a CO2 incubator for 24 h (culture conditions: 37°C, 5% CO2). After 24 h of incubation, the medium was removed, and freshly prepared medium containing different concentrations of 6TCl-PS NPs was added. After 12 h of incubation, the cells were washed three times with PBS buffer, and 10 μL of MTT solution (5 mg / mL) was added to each well. The cells were then incubated for another 4 h, after which the medium was removed, 100 μL of DMSO was added to each well, and the cells were shaken for 10 min. Finally, the absorbance at 490 nm was measured using a microplate reader, and the values ​​were recorded. Cell viability was determined by formula 7: (Equation 7) Calculate cytotoxicity ( ),in , The absorbance values ​​are for the experimental group and the control group, respectively. Six groups were measured in parallel for each concentration.

[0037] 8. Mouse imaging Female Kunming rats aged 4-5 weeks were purchased for imaging experiments. All animal experiments in this study complied with the requirements of the Animal Ethics Committee of Northwestern Polytechnical University. Mouse breast cancer 4T1 cells were pre-cultured in an incubator at 37°C and 5% CO2 using RPMI-1640 medium (10% fetal bovine serum, 100 μg / mL penicillin and 0.1 mg / mL streptomycin).

[0038] (1) Tumor modeling Observe the growth of 4T1 cells under a microscope. When the cells in the culture flask have good morphology, clear outlines, and a growth density of approximately 90%, digest the cells with trypsin, centrifuge, and resuspend them in pre-chilled PBS, adjusting the cell concentration in the PBS to 1×10⁻⁶. 7 After the cell suspension was reduced to a density of 2 × 10⁶ cells / mL, it was temporarily stored in an ice box. Hair on the backs of mice requiring tumor implantation was removed using a hair removal device and depilatory cream, with the tumor implantation site chosen on the right side of the mouse's back. Before inoculation, the cell suspension was thoroughly agitated and the injection site was disinfected with an alcohol swab. Subsequently, 200 μL of cell suspension (containing 2 × 10⁶ cells / mL) was subcutaneously injected into each mouse. 6(4T1 tumor cells): Before inoculation, expel all air from the syringe. Insert the needle at a 45-degree angle to the mouse's skin, then push it in parallel for about 1 cm before slowly injecting. After injection, slide the needle subcutaneously several times to help the cells clump together and reduce leakage of cell suspension from the needle puncture site. After cell inoculation, return the mice to their cages and observe the growth of the tumor at the inoculation site daily. Weigh the mice and record the tumor volume using calipers. Continue inoculation until the volume reaches 80 mm². 3 Subsequent tumor imaging studies will be conducted. The tumor volume calculation formula is as follows (Equation 8): (Equation 8) (2) Vascular imaging Healthy and vigorous normal mice were selected for whole-body vascular imaging. Before imaging, hair was removed from the abdomen and limbs of the mice using a hair removal device and depilatory cream. An appropriate amount of 6TCl-PS NPs concentrate was diluted with PBS to a concentration of 250 μg / mL. 200 μL of the sample solution was injected into the tail vein of each mouse using an insulin syringe. After tail vein administration, the mice were placed back-up in the center of the imaging stage, and the vascular system was immediately imaged using a near-infrared II in vivo imaging system. An 808 nm laser was used to excite fluorescence, and the instrument parameters were set to a 1000 nm long-pass filter and a 5 ms exposure time (1000 nm LP, 5 ms). Vascular images were acquired every 30 s, with a total imaging time of 5 minutes. During the imaging process, isoflurane was introduced into the anesthesia device to anesthetize the mice. The mice's vital signs were constantly monitored, and the isoflurane gas flow rate and anesthesia time were adjusted as needed to maintain the mice's viability. An vascular imaging result was acquired in parallel from three healthy mice under the same growth conditions, with n = 3.

[0039] (3) Lymphoma Healthy and vigorous normal mice were selected for hind limb lymphatic imaging. Before imaging, hair was removed from the back of both hind limbs using a hair removal device and depilatory cream. An appropriate amount of concentrated 6TCl-PS NPs stock solution was diluted with PBS to a concentration of 250 μg / mL. Simultaneously, an appropriate amount of commercial dye ICG was prepared as a 250 μg / mL PBS dilution for the control experiment. Using an insulin syringe, 20 μL of 6TCl-PS NPs (250 μg / mL) was injected subcutaneously into the right hind paw of the mouse. The needle was inserted subcutaneously at a 15-degree angle upwards from the center of the paw, advancing approximately 1 cm before slow injection. After injection, the needle was withdrawn, and gentle pressure was applied to ensure smooth entry of the solution into the lymphatic tissue. Additionally, using an insulin syringe, 20 μL of ICG solution (250 μg / mL) was injected subcutaneously into the left hind paw of the same mouse as a control experiment. 20 μL was injected into each hind paw pad of each mouse. After subcutaneous administration to the paw pads, 30 minutes were allowed to ensure sufficient diffusion of the material through lymphatic circulation. Subsequently, the mice were placed back-up in the center of the imaging stage, and their lymph nodes and lymphatic vessels were imaged using a near-infrared II in vivo imaging system. An 808 nm laser was used to excite fluorescence, and the instrument parameters were set to a 1000 nm long-pass filter and a 5 ms exposure time (1000 nm LP, 5 ms). Images were acquired every 10 minutes for one hour. During the imaging process, isoflurane was introduced into the anesthesia device to anesthetize the mice, and their vital signs were constantly monitored. The isoflurane gas flow rate and anesthesia time were adjusted as needed to maintain the mice's viability. Lymphatic imaging results were acquired in parallel from three healthy mice under the same growth conditions, with n = 3.

[0040] (4) Tumor imaging Select a tumor with a volume of approximately 80 mm. 3 Healthy tumor-bearing mice were used for tumor imaging experiments. Before imaging, hair was removed from the tumor area using a hair removal device and depilatory cream. An appropriate amount of concentrated 6TCl-PS NPs stock solution was diluted with PBS to a concentration of 250 μg / mL, and each mouse was injected intravenously via the tail vein (200 μL, 250 μg / mL). Timing began at the end of the tail vein administration, and images of tumor-bearing mice were acquired using a near-infrared II in vivo imaging system at time points of 3, 6, 9, 12, 24, 36, 48, 72, and 120 h. Imaging conditions (λ...)... ex = 808 nm, 1000 nm LP, 5 ms). During the imaging process, mice were anesthetized only at the imaging time point, and the isoflurane gas flow rate and anesthesia time were adjusted in a timely manner to maintain the vitality of the mice. With n=3, tumor imaging results were acquired in parallel from three tumor-bearing mice under the same growth conditions.

[0041] (5) Imaging of ex vivo organs Real-time quantitative analysis of in vivo imaging results in mice. When the fluorescence signal in the tumor area of ​​the mouse began to decrease, the mouse was immediately euthanized by cervical dislocation. Its heart, liver, spleen, lungs, kidneys, and tumor were dissected, collected, and temporarily stored in paraformaldehyde. Subsequently, under the same imaging conditions (λ... ex = 808 nm, 1000 nm LP, 5 ms), remove the organs and tumors of the mouse and place them in a clean culture dish. Use a near-infrared spectroscopy system to acquire fluorescence images of the isolated mouse organs.

[0042] The experimental and testing results are analyzed as follows: 1. Molecular structure characterization Molecular structures were characterized using proton nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. For example... Figure 3 As shown, 1 1H NMR (500 MHz, CDCl3, ppm): δ = 8.84 (s, 2H), 8.73 (s, 2H), 8.12 (s, 2H), 7.93 (s, 2H), 7.18 (s, 16H), 2.60–2.54 (m, 8H), 1.59 (p, 8H), 1.37–1.30 (m, 8H), 1.30–1.25 (m, 16H), 0.87–0.83 (t, 12H). The 1H NMR data of the 6TCl molecule showed a high agreement with the predicted values, confirming the successful synthesis of the target product.

[0043] like Figure 4 As shown, the mass spectrometry results for Waters Xevo GXS TOF (ESI): C 94 H 76 Cl4N4O2S6(M + Theoretical value: 1632.4128; Measured value: 1632.4091 (Error: -2.26 ppm). This proves the successful synthesis of the target product.

[0044] 2. Investigation of molecular fluorescence properties in different solvents First, the ground-state (S0) geometry of the 6TCl molecule was studied at the RB3LYP 6-31 G (D, P) level, and optimized using density functional theory (DFT). Figure 5 As shown in figure a, the optimized ground state 6TCl exhibits a planar configuration. This configuration suppresses the free rotation of the molecule in space, which is beneficial for reducing energy loss during nonradiative transitions. The calculated electrostatic potential (ESP) of 6TCl is shown in the figure. Figure 5(b) indicates that the negative charge is mainly concentrated on the nitrogen atom of the cyano group and the oxygen atom of the carbonyl group, while the positive charge is mainly concentrated near the thiophene ring and the alkyl chain, exhibiting typical characteristics of molecules based on a donor (D)-acceptor (A) structure. Furthermore, the calculated HOMO and LUMO energy levels are -5.37 eV and -3.59 eV, respectively, with a band gap of 1.78 eV. Figure 5 c).

[0045] To investigate the fluorescence properties of 6TCl in different solvents and to ensure that the absorbance of each solution at 808 nm was consistent, the absorption and emission spectra of 6TCl in toluene, tetrahydrofuran, and n-hexane were measured using a UV-Vis spectrophotometer and a steady-state-transient fluorescence spectrometer. Figure 6 Subsequently, under the same imaging conditions (λ) ex Dark-field fluorescence imaging of the above three solutions, each with a volume of 1 mL, was performed using a near-infrared II imager at 808 nm, 1000 nm LP, and 5 ms.

[0046] The results showed that 6TCl has strong absorption in the visible light region and the NIR-I region (600-850 nm), with the maximum absorption peak at 770 nm. Figure 7 a). Its maximum emission peak in the NIR-II region is located at 930 nm, and the tail peak can reach over 1100 nm. Figure 7 a). The absorbance of 6TCl at 808 nm in toluene (TOL), tetrahydrofuran (THF), and n-hexane (HE) was adjusted to the same level of 0.41. The fluorescence spectra of the three solvents under 808 nm laser excitation showed that 6TCl exhibited the strongest fluorescence emission in TOL. Figure 7 b). Regarding Figure 7 Quantitative analysis of dark-field imaging results of the same volume of solution in b under 808 nm laser showed that the average fluorescence intensity of 6TCl in TOL was more than 2.75 times and 100 times that in THF and HE, respectively. Figure 7 c). The above data indicate that 6TCl exhibits near-infrared II fluorescence emission in organic solvents, demonstrating potential for near-infrared II fluorescence imaging. Furthermore, 6TCl shows the highest fluorescence intensity in toluene, suggesting that the toluene solvent environment may be beneficial for optimizing its fluorescence performance. However, the microscopic mechanism underlying the significant differences in fluorescence intensity of small molecules in different solvents remains unclear, and further analysis using excited-state dynamics techniques such as ultrafast spectroscopy is needed to elucidate its intrinsic mechanism at the molecular level.

[0047] 3. Excited state dynamics research To investigate the underlying mechanism of the significant differences in fluorescence intensity of 6TCl in different solvents, ultrafast transient absorption spectroscopy was used to directly observe the excited-state dynamics of 6TCl in different solvents. Due to the weak fluorescence in n-hexane, the excited-state behavior of the molecule in n-hexane was not further investigated. Two-dimensional transient absorption contour plots in different solvents revealed significantly different ground-state bleaching signals. Figure 8 a, Figure 8 (b) This mainly reflects the non-radiative decay channel of excited-state energy dissipation. To quantitatively obtain decay kinetic information, a kinetic curve at a specific wavelength (991 nm) was extracted and globally fitted using a multi-exponential model. Figure 8 c). The fitting results show that in toluene, the excited-state kinetics of 6TCl exhibit two lifetime components: 19% τ1 = 152 ps and 81% τ2 = 1020 ps. In tetrahydrofuran, these are 72% τ1 = 22 ps and 28% τ2 = 335 ps. Both lifetime components in THF are significantly shorter than their corresponding components in TOL.

[0048] By correlating with measurements showing stronger fluorescence in the TOL as indicated by steady-state spectroscopy, the two long-lived components (1020 ps in TOL and 335 ps in THF) were attributed to radiative decay processes of the molecules, i.e., fluorescence emission. After identifying the long-lived components as radiative decay, the solvent-dependent short-lived components (152 ps in TOL and 22 ps in THF) can be reasonably attributed to nonradiative relaxation processes. Notably, the short-lived component in THF (22 ps) is much smaller than the corresponding component in TOL (152 ps), indicating that in THF, excited-state energy is rapidly dissipated through nonradiative pathways. Two-dimensional spectra provide more intuitive evidence: in THF, the excited-state absorption signal decays rapidly and has a narrow distribution within the initial delay time; while in TOL, the signal distribution is wider and persists over a longer timescale. Figure 8 a, Figure 8 (b) The above results reveal the potential mechanism by which the small molecule 6TCl exhibits maximum fluorescence intensity in toluene: the toluene environment effectively suppresses the nonradiative decay rate and stabilizes the excited state, thereby prolonging the radiative decay lifetime and enhancing fluorescence performance. This also provides key insights for optimizing material properties by constructing toluene-like microenvironments.

[0049] 4. Nanoparticle design concept Based on the excellent fluorescence properties and excited-state kinetics of 6TCl in toluene (TOL) solvent, we envision simulating the TOL microenvironment within biocompatible nanoparticles with 6TCl as the hydrophobic core to amplify the fluorescence brightness of nanomaterials in aqueous media. To simultaneously impart good water solubility to the nanomaterials, we selected an amphiphilic polymer, PS, with a molecular backbone containing benzene rings and hydrophilic side chains of PEG. 2000 -PEG 2000 A water-dispersible 6TCl-PS NPs nanoprobe was constructed by loading 6TCl using a nanoprecipitation method. Figure 9 To systematically verify the fluorescence modulation efficiency of the benzene ring doping strategy, 6TCl-DSPE NPs (DSPE-PEG) were set up. 2000 (Coated) control system.

[0050] 5. Study on the photophysical properties of nanoparticles To further investigate the impact of benzene ring doping strategies on material properties, the photophysical properties of 6TCl-DSPE NPs and 6TCl-PS NPs were systematically characterized. Dynamic light scattering (DLS) results showed that 6TCl-PS NPs exhibited a uniform spherical morphology with a hydrated particle size of approximately 42 nm. Figure 10 a). This size falls within the optimal range (40-200 nm) for passive targeting of solid tumors via the EPR effect, which is beneficial for avoiding liver retention and enhancing tumor tissue penetration. The absorption and emission spectra of the two nanoparticle dispersions show that, compared to 6TCl, the co-assembled 6TCl NPs both exhibit an absorption shoulder peak at 820 nm, and the emission tail peak is extended from 1100 nm to 1200 nm. Figure 10 d, Figure 10 e). Adjusting the absorbance of both at 808 nm to the same level, 6TCl-PS NPs exhibited a significantly enhanced fluorescence signal compared to 6TCl-DSPE NPs under 808 nm excitation, consistent with the fluorescence imaging results under dark field conditions with an equal volume of solution. Figure 10 c, 10e). Further quantitative analysis of fluorescence images of 6TCl-DSPE NPs and 6TCl-PS NPs showed that the average fluorescence intensity of 6TCl-PS NPs was 4.3 times that of 6TCl-DSPE NPs (c, 10e). Figure 10 f).

[0051] Figure 11 In this context, a concentration OD is set for each solution. 808 nm=0.02, 0.04, 0.06, 0.08, 0.10. (a) Absorption spectrum of 6TCl-DSPE NPs with gradient absorption at 808 nm in water. (b) Corresponding fluorescence spectrum of 6TCl-DSPE NPs in water. (c) Curve of integral fluorescence intensity of 6TCl-DSPE NPs in water under 808 nm excitation as a function of absorbance. 1000 nm, (d) Absorption spectrum of 6TCl-PS NPs with gradient absorption at 808 nm in water. (e) Corresponding fluorescence spectrum of 6TCl-PS NPs in water. (f) Curve of integral fluorescence intensity of 6TCl-PS NPs in water under 808 nm excitation as a function of absorbance. 1000 nm, (g) Absorption spectrum of IR-1061 with gradient absorption at 808 nm in DCM. (h) Corresponding fluorescence spectrum of IR-1061 in DCM. (i) Curve of integral fluorescence intensity of IR-1061 under 808 nm excitation in DCM as a function of absorbance. 1000 nm, 1700 nm). From Figure 11 It can be seen that, using the commercial dye IR-1061 as a reference (QY: 0.59% in DCM), the fluorescence quantum yield of 6TCl-PS NPs in water was 4.6%, significantly higher than that of the control group (Φ). 6TCl-DSPE NPs Quantum yield ( = 1.3%) Figure 11 The above results confirm that, under consistent experimental conditions, the fluorescence enhancement of 6TCl-PS NPs is attributed to the TOL-like solvent microenvironment constructed within them. This verifies the feasibility and effectiveness of the benzene ring doping strategy proposed in this application for optimizing the fluorescence performance of 6TCl in aqueous media.

[0052] For the detailed process and data of calculating the fluorescence quantum yield of 6TCl-DSPE NPs and 6TCl-PS NPs, please refer to [link to relevant documentation]. Figure 11 Table 3. The absorption spectra of three solutions with absorbances at 808 nm of 0.02, 0.04, 0.06, 0.08, and 0.10 respectively—an aqueous solution of 6TCl-DSPENPs, an aqueous solution of 6TCl-PS NPs, and a dichloromethane solution of IR-1061—and their respective fluorescence spectra under 808 nm excitation were compared. The slope of the integral fluorescence spectrum-absorbance fitting curve was obtained. According to formula 6:

[0053] Calculate the fluorescence quantum yield of each of the two materials. Using the fluorescence quantum yield of IR-1061 in DCM (Φ = 0.59%) as a reference, calculate the refractive index η of different solvents. H2O = 1.33, η DCM = 1.42.

[0054] The parameters are recorded in Table 3. The fluorescence quantum yield Φ is calculated using the relative method. 6TCl-DSPE NPs = 1.3%, Φ 6TCl-PS NPs =4.6%, indicating that the benzene ring doping strategy can significantly enhance the fluorescence brightness of near-infrared II nanomaterials.

[0055] Table 3. Calculation of fluorescence quantum yield under benzene ring doping strategy

[0056] 6. Study on optimal nanoparticle construction parameters To further enhance the fluorescence brightness of 6TCl-PS NPs constructed based on a benzene ring doping strategy, this application adjusts the ratio (mi) of organic fluorescent molecules to amphiphilic polymer coating agents in the nanomaterials. 6TCl :m PS2000-PEG2000 This key construction parameter was finely controlled. Figure 12 a) In the process of preparing nanoparticles by nanoprecipitation, the organic molecules and the coating agent PS are modified. 2000 -PEG 2000 By adding different ratios, nanoparticles with varying benzene ring doping mass ratios of 1:10, 1:20, 1:50, 1:70, 1:100, and 1:200 were obtained. During the experiment, the following phenomena were observed: when the benzene ring doping ratio was further increased from 1:200 to 1:500, the nanoparticle solution exhibited a pearlescent color after sonication, and the concentrated solution after centrifugation was a viscous jelly-like consistency. It was speculated that the presence of excessive coating agent affected the water solubility of the nanoparticles, which is detrimental to further research and application. Therefore, the maximum mass ratio was set at 1:200.

[0057] Different mass ratios of 6TCl-PS NPs were adjusted to ensure they all exhibited the same absorption OD=0.79 at 808 nm. Figure 12 b), with PS 2000 -PEG 2000 With increasing dosage, the fluorescence signal of the 6TCl-PS nanomaterials gradually increased. It is worth noting that 6TCl-PS… 200 NPs compared to 6TCl-PS 100 The emission intensity of NPs increases dramatically ( Figure 12 c). Quantitative analysis of fluorescence intensity of different nanoparticle solutions under 808 nm excitation, 6TCl-PS 200The average fluorescence intensity of NPs reached 6 TCl-PS. 20 6.1 times that of NPs (Φ = 4.6%) Figure 12 d, Figure 12 e). Subsequently, to obtain more rigorous and comprehensive experimental conclusions, the fluorescence properties of the nanoparticle solutions at different mass ratios were measured again at the same concentration. For each component solution at 7 μg / mL ( Figure 10 f), 6TCl-PS 200 NPs' exhibited the strongest absorption and emission ( Figure 12 f, Figure 12 g), fluorescence imaging quantitative results of solutions with different mass ratios in the dark field showed that, at the same concentration, 6TCl-PS 200 The average fluorescence intensity of NPs can still reach 6TCl-PS. 20 5.6 times that of NPs'. Based on the above results, the mass ratio m 6TCl :m PS2000-PEG2000 6TCl-PS constructed under the condition of 1:200 200 NPs exhibit the best fluorescence amplification effect. Selecting this component of nanomaterials for subsequent research can significantly improve the detection sensitivity and image quality of NIR-II fluorescence imaging.

[0058] 7. Study on the universality of benzene ring doping strategy The effectiveness of the benzene ring doping strategy was verified through in-depth research on the fluorescence properties of 6TCl-PS NPs, a nanomaterial with the organic small molecule 6TCl as a hydrophobic core. To further investigate whether this strategy can positively regulate the fluorescence properties of nanomaterials constructed from organic molecules of different structures and whether it has universal value, three types of molecules—polymers (PF-TTQ) and conjugated small molecules (Y8, TTQ-NH)—were selected. Figure 13 As a representative organic molecule in terms of structure, it can be used to study the universality of benzene ring doping strategies.

[0059] Based on the nanoprecipitation method, benzene ring-doped nanoprobe models were constructed: PTTQ-PS NPs, Y8-PS NPs, and TQNH-PS NPs, and DSPE-PEG was set. 2000 The control group nanoparticles coated with PTTQ-DSPE NPs, Y8-DSPE NPs, and TQNH-DSPE NPs were used. Fluorescence properties were characterized by the near-infrared II fluorescence quantum yield of each nanomaterial. Figure 14 , Figure 15 , Figure 16 , Figure 17As shown in Tables 4 and 5. It should be noted that in the preliminary qualitative analysis, the polymer PF-TTQ molecules showed almost no absorption at 808 nm, making it difficult to excite NIR-II fluorescence signals at this wavelength. Therefore, for PTTQ-PSNPs and PTTQ-DSPE NPs, the excitation wavelength was set to 660 nm for subsequent detection.

[0060] The results showed that the fluorescence quantum yields of PTTQ-PS NPs, Y8-PS NPs, and TQNH-PS NPs doped with benzene rings in the nanostructure were 0.72%, 21.5%, and 1.90%, respectively. Compared with the corresponding benzene-ring-free materials PTTQ-DSPE NPs (Φ1 = 0.31%), Y8-DSPE NPs (Φ2 = 19.5%), and TQNH-DSPE NPs (Φ3 = 1.04%), these yields were increased by 2.32 times, 1.10 times, and 1.83 times, respectively (Table 4). Figure 14 This indicates that, except for 6TCl molecules, doping organic molecules with different chemical structures with benzene rings in nanomaterials using them as a hydrophobic core can significantly enhance NIR-II fluorescence brightness. These results further demonstrate the good universality of the benzene ring doping strategy proposed in this study, providing a general, reliable, and scalable design approach for constructing high-brightness NIR-II organic nanomaterials.

[0061] Table 4. Generality Exploration - Fluorescence Quantum Yield Calculation

[0062] 8. Optimal in vitro characterization of nanoparticles To evaluate the optimal quality ratio of 6TCl-PS 200 The in vivo applicability potential of NPs was assessed by characterizing the hydration dynamics and surface morphology of the materials using DLS and TEM to determine whether the materials possess the sizing basis for long-term in vivo circulation and passive accumulation at tumor sites. 6TCl-PS was calculated. 200 The fluorescence quantum yield of NPs in water was determined to evaluate their fluorescence properties in detail, and the fluorescence quantum yield of NPs after 6TCl-PS was measured by the MTT assay. 200 Cell viability of MC3T3 cells after incubation with NPs was used to evaluate the cytotoxicity of the material.

[0063] Transmission electron microscopy (TEM) and dynamic light scattering (DLS) results indicate that 6TCl-PS 200 The NPs exhibit a uniform spherical morphology with a hydration dynamic size of 84.05 nm, making them suitable for in vivo tumor imaging via enhanced permeability and retention (EPR) effects. Figure 18The polydispersity index (PDI) reflects the degree of dispersion of nanomaterials. In an aqueous environment, 6TCl-PS... 200 The polydispersity coefficient of NPs is only 0.255, indicating that 6TCl-PS 200 NPs have good dispersibility, which helps maintain the stability of nanoparticles in physiological environments.

[0064] Using the fluorescence quantum yield of IR-1061 in DCM (Φ = 0.59%) as a reference, 6TCl-PS was calculated according to Equation 6. 200 Fluorescence quantum yield of NPs ( Figure 19 Data shows that 6TCl-PS 200 The NPs exhibited a high fluorescence quantum yield in water of up to 40%, representing a 135-fold enhancement compared to the initial 6TCl-DSPE NPs. 6TCl-PS 200 The NPs exhibit surprising fluorescence intensity in water, and their superior fluorescence performance will significantly improve imaging quality and resolution.

[0065] With different concentrations of 6TCl-PS 200 After co-incubation of NPs in α-MEM medium for 12 h, the cell viability level of mouse embryonic osteoblast MC3T3 cells was as follows: Figure 20 As shown in the figure. The results showed that cell viability remained above 90% within a concentration range of 20 μg / mL to 100 μg / mL. Even at the highest concentration (100 μg / mL), the cell viability of MC3T3 cells still reached 94%, showing no significant decrease compared to the control group without nanomaterial treatment, indicating that 6TCl-PS 200 NPs had no significant effect on normal cellular metabolic activities within the measured concentration range, exhibiting low cytotoxicity and good biocompatibility, demonstrating their potential for in vivo imaging applications.

[0066] 9. Imaging of blood vessels, lymph nodes, and tumors To evaluate 6TCl-PS 200 The in vivo NIR-II fluorescence imaging potential of NPs is highest at 6TCl-PS. 200 NPs-mediated imaging of blood vessels, lymph nodes, and tumors in mice.

[0067] Tail vein injection of 6TCl-PS 200 Immediately after NPs were placed in PBS solution (200 μL, 250 μg / mL), real-time imaging of the abdominal blood vessels of mice was performed using a near-infrared II in vivo imaging system. For example... Figure 21 As shown in figure a, blood vessels in the mouse abdomen are clearly visible under 808 nm laser irradiation. This is for preliminary evaluation of 6TCl-PS. 200NPs (NPs) vascular imaging resolution allows for quantitative analysis of fluorescence signals at the same cross-section within a specific vascular region, with the cross-section marked by a red line. Figure 21 a). Gaussian fitting was performed on the fluorescence intensity values ​​of blood vessels in different locations ( Figure 21 b) Imaging resolution was quantified using the full width at half maximum (FWHM). Ideally, a smaller FWHM value indicates higher imaging system resolution. Results showed that using a 1000 nm long-pass filter, the Gaussian fitting curve FWHM for mouse vascular fluorescence signals was 0.37 mm, indicating that 6TCl-PS... 200 NPs enable high-resolution vascular imaging. Further quantification of fluorescence signals in the same area of ​​the vascular region and the background region of the image to analyze the signal-to-background ratio (SBR) showed an SBR of 7.80, indicating high contrast and good imaging quality. These results collectively demonstrate that under low-power laser conditions (1 W / cm²) and short exposure time (5 ms), injecting only 6TCl-PS... 200 High-quality real-time imaging of abdominal blood vessels can be achieved with NPs (200 μL, 250 μg / mL), which minimizes potential photodamage to skin tissue and demonstrates the application potential of this nanomaterial in real-time dynamic observation of blood vessels.

[0068] 6TCl-PS200 NPs in PBS solution (20 μL, 250 μg / mL) were subcutaneously injected into the right hind paw of mice, and an equal volume and concentration of ICG solution was injected into the left hind paw as a control. Figure 21 c). Thirty minutes after injection, near-infrared spectroscopy (NIRS) images of mouse hind limb lymph nodes were acquired using a near-infrared spectroscopy (NIRS) in vivo imaging system. The images showed that during the 40-minute imaging process, under the same imaging conditions (1 W / cm², 1000 nm LP, 5 ms), bright globular lymph nodes and slender lymphatic vessels were consistently observed in the right hind limb, and the fluorescence signal of 6TCl-PS200 NPs at the lymph nodes continuously increased with the extension of imaging time. In contrast, only weak fluorescence was observed at the lymph nodes in the left hind limb, and the ICG fluorescence signal in the left hind limb significantly decreased after 50 minutes. Figure 21 e). The above results indicate that, compared with the clinically commonly used dye ICG, 6TCl-PS200 NPs not only possess higher fluorescence intensity but also exhibit superior in vivo retention and circulatory stability. Quantitative analysis of lymph node fluorescence signals showed that the half-width at half-maximum (FWHM) of its cross-sectional intensity distribution was 0.35 mm (e). Figure 21 d) The signal-to-background ratio (SBR) of the lymphatic region to the background reaches 9.52. These data demonstrate that the 6TCl-PS200 NPs offer both high resolution and high contrast in lymphatic imaging, providing an effective tool for long-term, clear imaging of in vivo lymphatic systems.

[0069] 6TCl-PS injected into the tail vein of 4T1 tumor-bearing mice 200 NPs were in PBS solution (200 μL, 250 μg / mL), and mice were then subjected to NIR-II fluorescence imaging at different time points. Figure 22 a). Benefiting from the enhanced permeability and retention (EPR) effect, 6TCl-PS 200 NPs continuously accumulate at the tumor site, and the fluorescence signal in the tumor area gradually increases over time, reaching a peak at 36 hours after injection. Figure 22 b). In 6TCl-PS 200 In NPs-mediated NIR-II fluorescence imaging, the tumor outline is clearly visible; therefore, 36 hours post-injection is the optimal time point for guiding surgical resection of the tumor. 144 hours post-injection, in vitro imaging of mouse tumor tissue and major organs such as the heart, liver, spleen, lungs, and kidneys was performed. Figure 22 c). The results showed that strong fluorescence signals were observed only in tumor tissue and the liver, while weak fluorescence signals were observed in other organs, indicating that 6TCl-PS... 200 NPs can accumulate well at the tumor site and are partially metabolized by the liver after intravenous injection. These results demonstrate that 6TCl-PS... 200 NPs are efficiently enriched at tumor sites through a passive targeting mechanism and have excellent fluorescence imaging contrast within the NIR-II window, which can clearly show tumor boundaries, demonstrating great clinical application value in the fields of precision tumor imaging and surgical navigation.

[0070] 10. Biosafety Assessment To evaluate 6TCl-PS 200 The biosafety of NPs was assessed through H&E staining and pathological analysis of major mouse organs. For example... Figure 23 As shown, compared with the control group that was injected with PBS buffer alone, the experimental group mice showed no obvious pathological signs such as cell infiltration, deformation, or necrosis in major organs such as the heart, liver, spleen, lungs, and kidneys, indicating that the 6TCl-PS material... 200 NPs exhibit excellent biocompatibility in vivo and show no significant observable cytotoxicity, meeting the basic biosafety requirements for the clinical translation of nanoprobes.

[0071] In summary, this invention proposes a simple, efficient, and universal benzene ring doping strategy to simulate a toluene solvent atmosphere inside conjugated small molecule-based nanoparticles to enhance their fluorescence, thereby improving NIR-II fluorescence imaging.

[0072] (1) The fluorescence properties of the small molecule 6TCl in different solvents were analyzed, and the results showed that it had the highest fluorescence intensity in toluene. Further analysis of excited-state dynamics using ultrafast spectroscopy revealed the intrinsic mechanism of fluorescence enhancement in toluene: the hydrophobic and planar rigid benzene ring in toluene can regulate the molecular stacking mode and aggregation environment inside the nanostructure, thereby reducing fluorescence quenching caused by aggregation and prolonging the lifetime of radiative transition components.

[0073] (2) Based on the experimental results showing that the small molecule 6TCl exhibits the strongest fluorescence in toluene, a benzene ring doping strategy was proposed. The fluorescence intensity of 6TCl-PS NPs constructed based on this strategy can reach 4.3 times that of 6TCl-DSPE NPs without benzene ring doping. In addition, three organic molecules with different chemical structures were used to construct benzene ring-doped nanoparticle models. Compared with the corresponding materials without benzene rings, the fluorescence intensity of all of them was significantly enhanced, demonstrating the universality and effectiveness of the strategy.

[0074] (3) Optimization of small molecule 6TCl and coating agent PS in 6TCl-PS NPs nanostructure 2000 -PEG 2000 6TCl-PS was obtained by mass ratio 200 NPs. 6TCl-PS 200 The NPs exhibit a fluorescence quantum yield of up to 40% in water and demonstrate good biocompatibility. They can achieve prolonged in vivo circulation and can be efficiently enriched at tumor sites through enhanced permeation and retention (EPR) effects. They also exhibit good performance under low-power laser conditions (1 W / cm²). 2 With a short exposure time (5 ms), high signal-to-noise ratio and high resolution fluorescence imaging of mouse blood vessels, lymph nodes and tumors can be achieved using NIR-II.

[0075] The benzene ring doping strategy proposed in this invention provides a new method for developing high-brightness, highly biocompatible NIR-II organic nanomaterials, and the constructed 6TCl-PS 200 NPs provide high-brightness, highly biocompatible candidate materials with clinical translational potential for precise imaging of deep tissues, early tumor monitoring, and intraoperative navigation.

[0076] It should be noted that the names of the compounds in this application are as follows: 6TCl: 2,2'-[[6,6,12,12-tetrakis(4-octylphenyl)-6,12-dihydrothieno[2'',3'':4',5']thieno[3',2':4,5]cyclopenta[1,2-b]thieno[2''',3''':4'',5'']thieno[2'',3'':3',4']cyclopenta[1',2':4,5]thieno[2,3-d]thiophene-2,7-diyl]bis[methylidyne(5,6-dichloro-3-oxo-1H-indene-2,1(3H)-diylidene)]]bis-propanedinitrile PF-TTQ: 6,7-bis(4-(hexyloxy)phenyl)-4-(5-(7-methyl-9,9-dioctyl-9H-fluoren-2-yl)thiophen-2-yl)-9-(5-methylthiophen-2-yl)-[1,2,5]thiadiazolo[3,4-g]quinoxaline Y8: 2,2'-((5Z,5'Z)-((12,13-bis(2-ethylhexyl)-3,9-diundecyl-12,13-dihydro-[1,2,5]thiadiazolo[3,4-e]thieno[2'',3'':4',5']thieno[2',3':4,5]pyrrolo[3,2-g]thieno[2',3':4,5]thieno[3,2-b]indole-2,10-diyl)bis(methaneylylidene))bis(6-oxo-5,6-dihydro-4H-cyclopenta[c]thiophene-5,4-diylidene))dimalononitrile TTQ-NH: 6,6',6'',6'''-(((6,7-bis(4-(hexyloxy)phenyl)-[1,2,5]thiadiazolo[3,4-g]quinoxaline-4,9-diyl)bis(thiophene-5,2-diyl))bis(9H-fluorene-2,9,9-triyl))tetrakis(hexan-1-amine) In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0077] Other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the appended claims.

Claims

1. An organic nanomaterial, characterized in that, The organic nanomaterials are: 6TCl-PS NPs, PTTQ-PS NPs, Y8-PS NPs or TQNH-PS NPs.

2. The method for preparing organic nanomaterials as described in claim 1, characterized in that, The preparation method includes: S1, Dissolve the solid organic molecule in tetrahydrofuran to obtain a molecular stock solution, wherein the organic molecule is 6TCl, PF-TTQ, Y8 or TTQ-NH; S2, PS 2000 -PEG 2000 The solid was dissolved in tetrahydrofuran to obtain a coating agent stock solution; S3. Mix the molecular stock solution and the coating agent stock solution evenly, then add pure water to remove tetrahydrofuran from the solution. After filtration, a nanoparticle solution is obtained, which is an organic nanomaterial.

3. The method for preparing organic nanomaterials according to claim 2, characterized in that, In S1, the concentration of the molecular stock solution is 1 mg / mL.

4. The method for preparing organic nanomaterials according to claim 2, characterized in that, In S2, the concentration of the coating agent stock solution is 5 mg / mL.

5. The method for preparing organic nanomaterials according to claim 2, characterized in that, In S3, the volume ratio of the mixture consisting of the molecular stock solution and the coating agent stock solution to pure water is 1:

9.

6. The method for preparing organic nanomaterials according to claim 2, characterized in that, In S3, the solution was filtered three times using a 0.22 μm filter membrane.

7. An application of an organic nanomaterial, characterized in that, The organic nanomaterial of claim 1 is used for the fabrication of nanoprobes.

8. The application of the organic nanomaterial according to claim 7, characterized in that, The nanoprobes are used for in vivo imaging.

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