Organic nanoparticles, methods of preparation and use

Abstract

This invention relates to an organic nanoparticle, its preparation method, and its application. The organic nanoparticle is 4SBZ-F NPs, 6SBZ-F NPs, 4STP-F NPs, or 6STP-F NPs. The organic nanoparticles of this invention exhibit excellent NIR-II fluorescence brightness, good biocompatibility, and significantly improved photostability compared to the commercial dye ICG. They can be efficiently enriched at tumor sites through enhanced penetration and retention effects. High signal-to-noise ratio and high-resolution NIR-II fluorescence imaging of mouse blood vessels, lymph nodes, and tumors can be achieved under low-power laser and short exposure time conditions.

Description

Technical Field

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

[0002] By physically co-assembling small molecules with benzene ring-containing coating agents via nanoprecipitation, a benzene ring-doped microenvironment was constructed within nanoparticles with organic molecules as hydrophobic cores, effectively enhancing the fluorescence brightness of organic nanomaterials. However, this did not alter the intrinsic photophysical properties of the molecules.

[0003] In the design of organic fluorescent molecules, strategies to enhance luminescence performance through structural modification have been widely reported. In July 2024, the research team of Ben-Chung Tang at the Hong Kong University of Science and Technology, based on a DAD structure molecule constructed with triphenylamine (TPA) as the electron donor, benzobisthiadiazole (BBTD) as the electron acceptor, and 3,4-ethylenedioxythiophene (EDOT) as the conjugated bridging unit, introduced alkylthio group side chains into the thiophene bridging unit. Through the strong interaction between sulfur and nitrogen atoms, an intramolecular interlocking structure was formed to inhibit the intrinsic molecular motion, ultimately synthesizing the high-brightness fluorophore TSEH. TSEH exhibited a fluorescence emission peak of 1050 nm in THF. Compared with the other two TH molecules and TEH molecules containing only one pair of sulfur-nitrogen electrostatic locks (without / with only one pair of sulfur-nitrogen electrostatic locks), TESH, containing two pairs of sulfur-nitrogen electrostatic locks, showed the highest QY in water: 1.22%. TESH-mediated in vivo imaging of blood vessels and tumors, as well as in vitro biosensing based on antigen detection, were successfully achieved. In November 2024, Professor Wang Jianguo's research group at Inner Mongolia University proposed using the spatial conjugation of side-chain phenyl isomerization to construct three groups of ADA-type NIR-II isomers that simultaneously enhance fluorescence brightness and photothermal properties: o-series fluorophores with phenyl groups on the outer side of the side chain (o-ITNP, o-ITCT, o-IDTCT) and i-series fluorophores with phenyl groups on the inner side of the side chain (i-ITNP, i-ITCT, i-IDTCT). Compared to the weak CH-π interaction between the phenyl group and the interlayer molecular backbone in the i-series, the o-series establishes an extended interlocking plane in the monolayer with stronger π-π interactions, exhibiting enhanced fluorescence brightness and photothermal properties under 808 nm excitation. These are synthesized using the amphiphilic polymer DSPE-mPEG. 2000After coating, o-ITNP NPs with a QY of 5.6% and a PCE of 76% exhibited the best overall performance and were successfully applied to high-resolution near-infrared angiography and image-guided photothermal therapy for breast tumors. In February 2025, Hua Jianli's team at East China University of Science and Technology reported a novel multidimensional donor engineering strategy that can simultaneously modulate the molecular properties of NIR-II AIE at multiple levels of molecular, aggregate, and solvent interactions. The research team designed and synthesized a novel strong donor unit, diphenylamine indono[1,2-B]thiophene, and simultaneously introduced methoxy and 2,4,4-trimethylpentane-2-yl groups to regulate its properties. By coupling different donor units with thienylbenzothiadiazole to form three small DAD molecules, MPITBT, DPITBT, and OPITBT, we successfully developed OPITBT-RNPs, which have both high QY (1.1%) and good PCE (28.8%) in aqueous solution. In the nano-aggregate state, they showed significant type I ROS generation ability and outstanding multimodal phototherapy diagnostic performance. PDT / PTT synergistic therapy guided by NIR-II FLI / PAI / PTI effectively eradicated 4T1 tumors in situ in mice.

[0004] The aforementioned technical solutions modify molecular side chains or side groups through element substitution and group substitution, demonstrating significant effectiveness in optimizing the brightness and even achieving multi-dimensional synergistic optimization of near-infrared organic small molecules. This strongly supports the unquestionable priority of molecular structure design in the selection of material performance optimization strategies. However, the improvement in fluorescence brightness achieved by these side chain modification strategies is relatively limited. Of particular note is the fact that currently clinically approved cyanide molecules generally suffer from poor stability, easily degrading due to light, heat, and biological environmental factors, severely restricting their long-term imaging and diagnostic applications. Therefore, there is a lack of efficient and concise molecular engineering strategies for constructing highly stable fluorescent materials.

[0005] Therefore, it is necessary to improve one or more of the problems existing in the above-mentioned related technical solutions.

[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 organic nanoparticles, a preparation method, and applications, 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 nanoparticle, wherein the organic nanoparticle is 4SBZ-F NPs, 6SBZ-F NPs, 4STP-F NPs or 6STP-F NPs.

[0009] The present invention further provides a method for preparing organic nanoparticles, the method comprising the following steps: S1, Dissolve the solid organic molecule in tetrahydrofuran to obtain a molecular stock solution, wherein the organic molecule is 4SBZ, 4STP, 6SBZ or 6STP; S2, F127 powder is dissolved in tetrahydrofuran to obtain a coating agent stock solution; S3. The molecular stock solution and the coating agent stock solution are mixed evenly, then injected into pure water, ultrasonically treated, and then tetrahydrofuran is removed. After filtration, organic nanoparticles are obtained.

[0010] In this invention, the organic molecule is prepared by Knoevenagel condensation reaction.

[0011] In this invention, in S1, the concentration of the molecular stock solution is 1 mg / mL.

[0012] In this invention, in step S2, the concentration of the coating agent stock solution is 5 mg / mL.

[0013] In this invention, when the molecular stock solution and the coating agent stock solution are mixed, the mass ratio of organic molecules to coating agent is 1:20.

[0014] In this invention, in S3, the volume ratio of the mixture of molecular stock solution and coating agent stock solution to pure water is 1:9.

[0015] The present invention also provides an application of organic nanoparticles for bioimaging.

[0016] The technical solution provided by this invention may include the following beneficial effects: The organic nanoparticles of this invention exhibit excellent NIR-II fluorescence brightness, good biocompatibility, and significantly improved photostability compared to the commercial dye ICG. They can be efficiently enriched at tumor sites through enhanced penetration and retention effects. High signal-to-noise ratio and high-resolution NIR-II fluorescence imaging of mouse blood vessels, lymph nodes, and tumors can be achieved under low-power laser and short exposure time conditions. 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 synthetic equations for thiophene and benzene ring molecules in this invention are shown. Figure 3 The Jacobensky energy level diagram is shown. Figure 4 The compound 4SBZ is shown. 1 H NMR spectrum; Figure 5 The compound 4STP is shown. 1 H NMR spectrum; Figure 6 The compound 6SBZ is shown. 1 H NMR spectrum; Figure 7 The compound 6STP is shown. 1 H NMR spectrum; Figure 8 (a) Ground-state geometry of RB3LYP 6-31 G (D, P) optimized for 4STP, 4SBZ, 6STP, and 6SBZ; (b) Frontier molecular orbitals of 4STP, 4SBZ, 6STP, and 6SBZ based on density functional theory (DFT). Figure 9 The calculated molecular electrostatic potential (ESP) results are shown: (a) 4STP; (b) 4SBZ; (c) 6STP; (d) 6SBZ; Figure 10 (a) Absorption spectra of molecules 4SBZ, 4STP, 6SBZ, and 6STP in THF. (OD) 808 nm =0.1); (b) Fluorescence spectrum of solution a under 808 nm excitation; (c) Fluorescence imaging and quantitative results of 1 mL of the four solutions. Dark-field fluorescence images: (λ ex : 808 nm, 1000 nm LP, 20 ms); Figure 11 A schematic diagram of the preparation of 4SBZ, 4STP, 6SBZ, and 6STP nanoparticles is shown. Figure 12 The diagram shows the hydration dynamics, size, and surface morphology of the nanoparticles. Figure 13 (a) Absorption spectra of four nanoparticles in water (OD) 808 nm = 0.1); (b) Emission spectra of solutions with equal absorbance under 808 nm laser irradiation; (c) Bright-field and dark-field images and fluorescence quantum yield of 1 mL solutions with equal absorbance, dark-field image: (λ ex : 808 nm, 1000 nm LP, 20 ms); Figure 14The results of fluorescence quantum yield measurements for 4SBZ, 4STP, 6SBZ, and 6STP nanoparticles are shown. Figure 15 The photothermal conversion efficiency of 4SBZ, 4STP, 6SBZ, and 6STP nanomaterials is shown. Figure 16 The temperature change of the nanoparticle solution (500 μL, 25 μg / mL) during a complete heating-cooling cycle is shown, along with the linear relationship between temperature-dependent -Ln(θ) and time during cooling; laser irradiation time: 15 min, temperature cycle time: 30 min. (808 nm, 1 W / cm²) 2 ); (a) 6SBZ-F NPs solution; (b) 6STP-F NPs solution; (c) 4SBZ-F NPs solution; (d) 4STP-F NPs solution; Figure 17 (a) Bright-field and dark-field images and mean fluorescence intensity (λ) of IR-1061 and 4SBZ-F NPs (1 mL, 25 μg / mL) in ICG and DCM in water. ex 808 nm, 1 W / cm 2 (1000 nm LP, 20 ms); (b) Fluorescence intensity changes of ICG and 4SBZ-F NPs aqueous solutions under continuous laser irradiation for 30 min. (λ) ex 808nm, 1 W / cm 2 (1000 nm LP, 20 ms); Figure 18 The results show the effect of 4SBZ-F NPs on the cell viability of MC3T3 cells; Figure 19 (a) NIR-II imaging of whole-body blood vessels in mice (λ) ex 808 nm, 1 W / cm 2 (1000 nm LP, 30 ms); (b) Fitted curve of fluorescence intensity of blood vessel cross section in Figure a; (c) Local lymphocyte imaging in mice over a period of time (λ) ex 808 nm, 1 W / cm 2 (1000 nm LP, 30 ms); (d) Figure c: Fluorescence intensity fitting curve of lymph node cross section; Figure 20 (a) 4SBZ-F NPs-mediated mouse NIR-II tumor imaging at different time points (λ) ex 808 nm, 1 W / cm2 (1000 nm LP, 30 ms); (b) Changes in fluorescence intensity at the tumor site over time; (c) NIR-II fluorescence imaging images of isolated organs; (d) Quantitative analysis of fluorescence signals in isolated organs; Figure 21 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 focuses on the regulatory effect of terminal benzene ring cyclization on fluorescence behavior and proposes a novel "terminal benzene ring cyclization" brightness regulation strategy, aiming to provide new ideas for the design of high-performance near-infrared luminescent materials.

[0022] Accordingly, this invention designs and synthesizes two organic conjugated small molecule systems with near-infrared II fluorescence: the TP system (4STP, 6STP) with thiophene as the terminal group and the BZ system (4SBZ, 6SBZ) with benzene ring as the terminal group. The invention innovatively proposes a terminal benzene ringization strategy to improve the fluorescence performance of the materials and enhance their NIR-II imaging effect.

[0023] The names of the compounds are as follows: 4STP: 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 6STP: 2,2'-[[6,6,12,12-tetrakis(4-octylthiophen-2-yl)-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 4SBZ: 2,2'-((2Z,2'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(3-oxo-2,3-dihydro-1H-indene-2,1-diylidene))dimalononitrile 6SBZ: 2,2'-[[6,6,12,12-tetrakis(4-hexylphenyl)-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(3-oxo-1H-indene-2,1(3H)-diylidene)]]bis-propanedinitrile First, this application analyzed the fluorescence properties of different molecules in the same solvent (THF). The fluorescence intensity of the benzene ring-based molecules was increased by 1.60 times and 1.30 times compared to the corresponding thiophene-based molecules, preliminarily confirming that this strategy can effectively regulate the intrinsic fluorescence properties of molecules. Based on this, to further realize its application in biological systems, water-soluble nanoparticles were constructed using hydrophobic small molecules as the core. In the molecular aggregated state, the benzene ring-based nanoparticles still maintained a fluorescence enhancement of 1.70 times and 1.22 times compared to the corresponding thiophene-based nanoparticles, indicating that the introduction of terminal benzene rings can effectively achieve synergistic optimization of fluorescence brightness at the molecular and nanoscale levels.

[0024] Subsequently, from the perspective of excited-state energy relaxation, combined with complementary analysis of fluorescence and photothermal properties, the intrinsic mechanism of terminal benzene ringization enhancing fluorescence was elucidated in depth: introducing benzene rings as terminal groups into the DA-type conjugated framework, leveraging its structural rigidity to enhance molecular planarity, suppressing non-radiative energy dissipation caused by molecular vibration and rotation in the excited state, thereby improving radiative transition efficiency. The screened benzene ring-based nanoparticles, 4SBZ-F NPs, exhibited the best fluorescence performance (Φ = 2.51%), and compared to ICG which suffered severe photodegradation, they maintained over 85% of their initial brightness after 30 min of laser irradiation, demonstrating excellent photostability. They were successfully applied to high-brightness NIR-II vascular, lymphatic, and tumor imaging, such as... Figure 1 As shown.

[0025] The specific implementation method is as follows: 1. Experimental reagents Table 1 Main experimental materials and reagents

[0026] 2. Experimental apparatus Table 2 Experimental Instruments

[0027] 3. Organic molecule synthesis The organic molecule is 4SBZ, 4STP, 6SBZ or 6STP, all of which can be synthesized by Knoevenagel condensation reaction.

[0028] (1) Synthesis of 6SBZ, a benzene ring molecule like Figure 2 a) 6,6,12,12-tetra(4-hexylphenyl)-1,2-dihydro-thieno[2'',3'':4',5']thieno[3',2':4,5]cyclopentadieno[1,2-b]thieno[2',3':4,5]cyclopentadieno[1'',2'':4,5]thieno-2,8-dicarboxaldehyde (60 mg, 0.053 mmol) and 2-(oxo-2,dihydro-1H-inden-1-yl)malononitrile (102 mg, 0.53 mmol) were dissolved in anhydrous chloroform (10 mL) under an inert atmosphere after three nitrogen purgings. Pyridine (0.1 mL) was then slowly added dropwise as a catalyst, and the reaction was carried out under constant temperature and magnetic stirring in an oil bath for 16 h. After the reaction was completed, the system was cooled to room temperature. The mixture was then washed with methanol (100 mL), filtered, and the residue on the filter paper was dissolved in chloroform. The solvent was removed by rotary evaporation, and the collected residue was purified by silica gel column chromatography with petroleum ether / CH2Cl2 (1:1, v:v) as the eluent. The target product was finally obtained as a dark green solid (50 mg, 64%).

[0029] (2) Synthesis of thiophene-based molecules 6STP like Figure 2 a) 6,6,12,12-tetra(4-hexylphenyl)-1,2-dihydro-thieno[2'',3'':4',5']thieno[3',2':4,5]cyclopentadieno[1,2-b]thieno[2',3':4,5]cyclopentadieno[1'',2'':4,5]thieno-2,8-dicarboxaldehyde (62 mg, 0.059 mmol) and 2-(6-oxo-5,6-dihydro-4H-cyclopentadieno[c]thieno-4-alkylene)malonitrile (120 mg, 0.59 mmol) were dissolved in anhydrous chloroform (10 mL) under an inert atmosphere after three nitrogen purgings. Pyridine (0.1 mL) was then slowly added dropwise as a catalyst, and the reaction was carried out under constant temperature and magnetic stirring in an oil bath for 16 h. After the reaction was completed, the system was cooled to room temperature. The mixture was then washed with methanol (100 mL), filtered, and the residue on the filter paper was dissolved in chloroform. The solvent was removed by rotary evaporation, and the collected residue was purified by silica gel column chromatography with petroleum ether / CH2Cl2 (1:1, v:v) as the eluent. The target product was finally obtained as a dark green solid (60 mg, 67%).

[0030] (3) Synthesis of 4SBZ, a benzene ring molecule like Figure 2 b. Under nitrogen protection, dry pyridine (1 mL) was added to an anhydrous chloroform solution (100 mL) of 12,1-bis(2-ethylhexyl)-3,9-di-n-undecyl-12,1-dihydro-[1,2,5]thiadiazo[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-dicarboxaldehyde (140 mg, 0.123 mmol) and 2-(oxo-2,dihydro-1H-inden-1-ylidene)malonitrile (130 mg, 0.67 mmol). Then, pyridine (0.1 mL) was slowly added dropwise as a catalyst, and the reaction was carried out under constant temperature and magnetic stirring in an oil bath for 16 h. After the reaction was complete, the system was cooled to room temperature. The mixture was then washed with methanol (100 mL), filtered, and the residue on the filter paper was dissolved in chloroform. The solvent was removed by rotary evaporation, and the collected residue was purified by silica gel column chromatography using petroleum ether / CH2Cl3 (1:3, v:3v). The target product was finally obtained as a dark blue solid (140 mg, 76.5%).

[0031] (4) Synthesis of thiophene-based molecules 4STP like Figure 2 b. Under nitrogen protection, dry pyridine (1 mL) was added to an anhydrous chloroform solution (100 mL) of 12,1-bis(2-ethylhexyl)-3,9-di-n-undecyl-12,1-dihydro-[1,2,5]thiadiazo[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-dicarboxaldehyde (156 mg, 0.150 mmol) and 2-(6-oxo-5,6-dihydro-4H-cyclopenta[c]thieno-4-alkylene)malonitrile (150 mg, 0.75 mmol). Pyridine (0.1 mL) was then slowly added dropwise as a catalyst. The reaction was carried out under constant temperature and magnetic stirring in an oil bath for 16 minutes. h. After the reaction was complete, the system was cooled to room temperature. The mixture was then washed with methanol (100 mL), filtered, and the residue on the filter paper was dissolved in chloroform. The solvent was removed by rotary evaporation, and the collected residue was purified by silica gel column chromatography with petroleum ether / CH2Cl3 (1:3, v:3v) as the eluent. The target product was finally obtained as a dark blue solid (165 mg, 78%).

[0032] 4. Molecular calculation methods 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.

[0033] All calculations were performed using the Gaussian 16 quantum chemistry software package. Data post-processing and visualization were performed using GaussView 6.0.

[0034] 5. Preparation of nanoparticles In this experiment, benzene ring nanoparticles 4SBZ-F NPs and 6SBZ-F NPs and thiophene nanoparticles 4STP-F NPs and 6STP-F NPs were prepared by nanoprecipitation method.

[0035] Accurately weigh 1 mg each of 4SBZ, 4STP, 6SBZ, and 6STP solids, and 40 mg of F127 powder using an electronic balance. Dissolve each in tetrahydrofuran to obtain four molecular stock solutions (1 mg / mL, 1 mL) and an F127 coating agent stock solution (5 mg / mL, 8 mL). Mix 400 μL of the molecular stock solution with 1.6 mL of the coating agent stock solution, ensuring a molecular to coating agent mass ratio of 1:20, and sonicate thoroughly. For the nanoprecipitation method, the mixture and pure water must have a volume ratio of 1:9. Take 18 mL of pure water, and under sonication conditions, rapidly inject 2 mL of the mixture into the pure water, continuing sonication for 40 minutes. After sonication, allow to stand overnight to allow the remaining tetrahydrofuran to completely evaporate. The following day, the solution was filtered 2-3 times through a 0.22 μm filter membrane, and the clear and transparent nanoparticle filtrate (4SBZ-F NPs, 4STP-F NPs, 6SBZ-F NPs, and 6STP-F NPs) was collected. The solution was then concentrated in an ultracentrifuge at 5000 rpm for 10 min, and the concentrate was stored in a refrigerator at 4°C for later use.

[0036] 6. Fluorescence performance test (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)... 2This 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.

[0037] 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.

[0038] (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. FThe 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... F Characterizing 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.

[0039] 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. .

[0040] 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.

[0041] 7. Photothermal performance test (1) Experimental principle Organic conjugated small molecules absorb photons under specific wavelengths of light, changing from their ground state. Transition to an excited state (usually an excited singlet state) The energy absorbed by molecules in the excited state is released mainly through two competing pathways: radiative relaxation and non-radiative relaxation. Figure 3 Radiative relaxation releases energy in the form of photons, manifesting as fluorescence emission; non-radiative relaxation, on the other hand, dissipates energy as heat through processes such as intramolecular vibrational relaxation, internal conversion, intersystem crossing, and subsequent thermalization, leading to an increase in system temperature and the generation of a photothermal effect. The temperature rise and heat flow detected in photothermal tests are essentially non-radiative heat dissipation; therefore, the photothermal response is closely related to the non-radiative relaxation process: the higher the proportion of non-radiative transitions, the more heat is generated per unit time, and the stronger the photothermal signal; conversely, if radiative transitions are more dominant (stronger fluorescence), the proportion of energy that can be converted into heat decreases, and the photothermal signal is relatively weakened.

[0042] Under steady-state or quasi-steady-state conditions, assuming no photochemical reaction occurs, the conservation of excited-state energy can be summarized as follows: (Equation 7) in The fluorescence quantum yield represents the proportion of radiation released. This represents the nonradiative dissipation ratio. Because the donor / acceptor strength, molecular planarity, conjugation length, intramolecular charge transfer, and aggregation state in the ADA molecular structure significantly affect the conformational relaxation and vibrational coupling strength of the excited state, thereby altering the nonradiative decay rate. With radiation attenuation rate Relative size: (Equation 8) Therefore, photothermal testing is equivalent to testing from the perspective of energy dissipation. To perform quantitative characterization that is "macroscopically observable".

[0043] Accordingly, this invention obtains the photothermal response temperature rise curve and photothermal conversion efficiency of conjugated small molecules under the same excitation conditions by conducting photothermal response tests, and compares them with the fluorescence characterization results. By comparing the differences in photothermal response of different molecules under the same test conditions, the dominant factors and underlying mechanisms of fluorescence performance differences are analyzed, thus completing the complementary verification of photothermal and fluorescence data.

[0044] (2) Test conditions Photothermal testing was conducted using an infrared thermal imager. This highly sensitive imager allowed for real-time and precise monitoring and recording of the temperature change curves and spatial distribution of the sample system under laser irradiation. Considering the material's absorption peak and the operating wavelength of mainstream lasers, an 808 nm laser was selected as the excitation wavelength for this experiment to determine the material's photothermal properties. The entire testing process was conducted in the dark, and the sample solution was stored at low temperature and in the dark.

[0045] (3) Experimental operation Take a small amount of nanoparticle stock solution and dilute it with pure water to prepare a 25 μg / mL solution as the test sample solution. Place 500 μL each of 4SBZ-F NPs, 4STP-F NPs, 6SBZ-F NPs, and 6STP-F NPs (25 μg / mL) into a 1 mL centrifuge tube (the smallest size available). Fix the tube vertically at the same height as the lens of a handheld thermal imager, approximately 15 cm away from the sample, ensuring the image is centered within the thermal imager. Secure the laser output end tightly against the side wall of the centrifuge tube to minimize heat loss, and set the laser power to 1 W / cm². 2 Data was acquired using a thermal imager (808 nm, 1 W / cm²). 2 Photothermal images of four nanoparticle solutions (500 μL, 25 μg / mL) under laser irradiation. Starting at time s, solution images were acquired every minute, recording complete heating-cooling cycles, with each cycle lasting 15 minutes. To evaluate the photothermal stability of the material, all samples underwent five consecutive thermal cycles independently under standard experimental conditions. Time-temperature curves were recorded throughout the experiment.

[0046] (4) Calculation of photothermal conversion efficiency After obtaining the temperature rise curve of the sample under continuous irradiation with a specific laser wavelength, its photothermal conversion efficiency is further calculated to quantitatively analyze the photothermal properties of different materials. This invention uses the classical model established by Roper et al. and widely adopted to calculate the photothermal conversion efficiency. (Equation 9).

[0047] (Equation 9) in, The total heat transfer coefficient from the system to the environment. For effective heat transfer area, The initial ambient temperature, This is the final steady-state temperature of the solution system during the heating process. For laser power, For solvent thermal power, The absorbance of the sample at a laser wavelength of 808 nm is given. Temperature changes of the sample solution before laser irradiation, during irradiation, and after the laser is turned off are recorded to obtain temperature-time curves. The steady-state temperature of the solution before irradiation was taken as the ambient temperature. The steady-state temperature that the solution eventually reaches after irradiation is taken as... The power of the 808 nm laser is set to 1 W / cm². 2 The absorbance of the sample at a specific wavelength was measured using a UV-Vis spectrophotometer. .

[0048] heat transfer coefficient product The calculation is as follows: When the system reaches thermal steady state, the light energy absorbed by the sample is dissipated as heat. For the cooling process after the laser is turned off, the thermodynamic process of the system at any given moment follows the following energy balance equation: (Equation 10) in, and These represent the mass of solute and solvent in the sample system and their corresponding specific heat capacities. Let be the system temperature at any given moment during the cooling process. In a dilute solution system with a single solute, Approximately the mass of the solution. Approximately the specific heat capacity of the solvent (aqueous phase), take Equation 10 simplifies to: (Equation 11) make Then we have Equation 12: (Equation 12) Let the time constant Integrating the above equation and taking its logarithm, we obtain equation 13: (Equation 13) Therefore, the temperature difference data between each moment of the cooling process and the initial temperature moment are extracted, and a logarithmic temperature difference-time fitting curve is plotted. The slope of the curve is... Then the heat transfer coefficient product .

[0049] Solvent thermal power Calculations: Under the same experimental conditions (808 nm, 1 W / cm²), 2The temperature rise and fall curves of 500 μL of pure solvent were tested over 30 min, and the ambient temperature was recorded. and According to Equation 10, the energy balance equation, .

[0050] Finally, the solutions of each nanomaterial... , , Laser power and the absorbance of the sample at 808 nm Substitute into Equation 9 to calculate the photothermal conversion efficiency of each material. .

[0051] 8. 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 4SBZ-F 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 calculated using formula 2-5, and six groups were measured in parallel for each concentration.

[0052] 9. 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).

[0053] (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. 7After 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 formula for calculating tumor volume is as follows:

[0054] (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 4SBZ-F NPs concentrated stock solution 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 for fluorescence excitation, and the instrument parameters were set to a 1000 nm long-pass filter and a 30 ms exposure time (808 nm, 1000 nm LP, 30 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.

[0055] (3) Lymphoma Healthy and vigorous normal mice were selected for hind limb lymphatic imaging. Before imaging, hair was removed from the right hind limb region of the mice using a hair removal device and depilatory cream. An appropriate amount of 4SBZ-F NPs concentrate was diluted with PBS to a concentration of 250 μg / mL. Using an insulin syringe, the diluted sample solution was injected subcutaneously into the right hind paw of each 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 facilitate the solution's entry into the lymphatic tissue. 20 μL was injected into the right hind paw pad of each mouse. After subcutaneous administration to the paw pad, 10 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 the mouse lymph nodes and lymphatic vessels were imaged using a near-infrared II in vivo imaging system. An 808 nm laser was used for fluorescence excitation, with instrument parameters set to a 1000 nm long-pass filter and an exposure time of 30 ms (808 nm, 1000 nm LP, 30 ms). Images were acquired every 10 minutes for 1.5 hours. 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 their vitality. Lymphatic imaging results were acquired in parallel from three healthy mice under the same growth conditions, with n = 3.

[0056] (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 4SBZ-F NPs stock solution was diluted with PBS to a concentration of 250 μg / mL, and each mouse was intravenously injected with 200 μL (250 μg / mL) of 4SBZ-F NPs via tail vein. Timing was started from the completion of tail vein administration, and images of tumor-bearing mice were acquired using a near-infrared II in vivo imaging system at time points of 0, 2, 4, 7, 20, 32, 44, 68, and 116 h. Imaging conditions (λ...)... ex = 808 nm, 1000 nm LP, 30 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.

[0057] (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, 30 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.

[0058] The experimental and testing results are analyzed as follows: 1. Molecular structure characterization The molecular structures of 4SBZ, 4STP, 6SBZ, and 6STP were characterized by proton nuclear magnetic resonance spectroscopy. Figure 4 As shown, 1 1H NMR (500 MHz, CDCl3, ppm): δ = 9.19 (s, 2H), 8.72 (d, 2H), 7.95 (d, 2H), 7.80–7.74 (t, 4H), 4.78 (d, 4H), 2.22–2.02 (m, 2H), 1.96–1.79 (m, 4H), 1.56–1.48 (m, 4H), 1.47–1.37 (m, 4H), 1.34–1.17 (m, 32H), 1.01 (t, 4H), 0.86 (t, 6H), 0.74 (dd, 6H), 0.65 (dd, 6H). The 1H NMR data of molecule 4SBZ showed a high degree of agreement with the predicted values, confirming the successful synthesis of the target product.

[0059] like Figure 5 As shown, 1 1H NMR (500 MHz, CDCl3, ppm): δ = 9.09 (s, 2H), 8.40 (d, 2H), 7.96 (d, 2H), 4.75 (d, 4H), 3.23 (t, 4H), 2.12–2.06 (m, 2H), 1.88 (p, 4H), 1.5–1.47 (m, 4H), 1.40–1.33 (m, 4H), 1.3–1.19 (m, 32H), 1.18–1.09 (m, 4H), 1.08–0.91 (m, 12H), 0.86 (t, 6H), 0.75 (t, 6H), 0.64 (t, 6H). The 1H NMR data of molecule 4STP showed a high agreement with the predicted values, confirming the successful synthesis of the target product.

[0060] like Figure 6 As shown, 11H NMR (500 MHz, CDCl3, ppm): δ = 8.84 (s, 2H), 8.66 (d, 2H), 8.12 (s, 2H), 7.90 (d, 2H), 7.78–7.69 (m, 4H), 7.18 (dd, 16H), 1.64–1.56 (m, 8H), 1.38–1.21 (m, 24H), 0.85 (t, 12H). The 1H NMR data of molecule 6SBZ showed a high agreement with the predicted values, confirming the successful synthesis of the target product.

[0061] like Figure 7 As shown, 1 1H NMR (500 MHz, CDCl3, ppm): δ = 8.84 (s, 2H), 8.66 (d, 2H), 8.12 (s, 2H), 7.90 (d, 2H), 7.78–7.69 (m, 4H), 7.18 (dd, 16H), 1.64–1.56 (m, 8H), 1.38–1.21 (m, 24H), 0.85 (t, 12H). The 1H NMR data of molecule 6STP showed good agreement with the predicted values, confirming the successful synthesis of the target product.

[0062] 2. Molecular structure optimization To explore the potential impact of different end-group structures on optical properties, density functional theory (DFT) was used to study the optimized ground state of the molecule at the RB3LYP 6-31 G (D, P) level. (Geometric structure, HOMO-LUMO orbital energy levels, and charge distribution.) Figure 8 As shown in Figure a, the optimized ground-state benzene ring molecules 4SBZ and 6SBZ exhibit higher coplanarity compared to the thiophene molecules 4STP and 6STP. This characteristic suppresses the nonradiative relaxation channels of excited states based on molecular vibrations and rotations, which is beneficial for enhancing molecular fluorescence. HOMO-LUMO energy level calculations show that ( Figure 8 (b) For the four conjugated molecules, the highest occupied molecular orbital (HOMO) is mainly located in the donor unit, while the lowest unoccupied molecular orbital (LUMO) is delocalized along the entire conjugated backbone. Compared with 4STP and 6STP corresponding to thiophene end groups, the benzene ring-terminated molecules 4SBZ and 6SBZ show narrower band gaps (1.82 eV, 1.87 eV), indicating that the introduction of benzene ring end groups can effectively expand the π-conjugated system of the molecule and enhance intramolecular charge transfer. The above calculation results predict the dual advantages of introducing a benzene ring into the molecular structure: firstly, the reduction of the optical band gap promotes a redshift in the absorption and emission spectra, thereby broadening the spectral response range; secondly, the improvement in planarity helps to enhance the radiative transition efficiency of the molecule, thereby enhancing the fluorescence emission intensity.

[0063] Furthermore, the charge distribution was analyzed by calculating the electrostatic potential (ESP) of each molecule. For example... Figure 9 As shown, the negative charges of the four molecules are mainly concentrated on the nitrogen atom of the cyano group and the oxygen atom of the carbonyl group. The positive charges of 4STP and 6STP are mainly concentrated around the alkyl chain and partially distributed on the hydrogen atoms of the thiophene ring; while the positive charges of 4SBZ and 6SBZ, in addition to the alkyl chain, are also distributed on the hydrogen atoms of the benzene ring, showing the typical charge distribution characteristics of this type of ADA-type conjugated small molecule. Compared with the thiophene terminal molecules 4STP and 6STP ( Figure 9 a, Figure 9 c), the positively charged region and charge density on the surface of benzene ring-terminated molecules 4SBZ and 6SBZ decrease ( Figure 9 b, Figure 9 c) indicates that the introduction of the benzene ring promotes the uniform delocalization of π electrons throughout the conjugated framework, effectively reducing the local positive charge on the molecular surface. This difference in charge distribution further proves that, compared to the thiophene end group, the benzene ring end group can provide a more uniform and extended electron supply to the conjugated system. This conclusion is consistent with the aforementioned calculation results of the molecular orbital gap and geometry.

[0064] 3. Characterization of molecular fluorescence properties To investigate the effect of terminal benzene cyclization on molecular fluorescence properties, four molecules—4SBZ, 6SBZ, 4STP, and 6STP—were dissolved in THF. Their absorption and fluorescence spectra were measured using a UV-Vis spectrophotometer and a steady-state-transient fluorescence spectrometer, respectively. Dark-field fluorescence images of 1 mL of the solution were captured under 808 nm laser excitation for quantitative analysis (λex = 808 nm, 1000 nm LP, 20 ms). Since low concentrations maximally suppress the self-absorption effect of the solution, the absorbance of the solution at the excitation wavelength (808 nm) was adjusted to a uniform 0.1 (λex = 808 nm, 1000 nm LP, 20 ms). Figure 10 a).

[0065] The results showed that all four molecules exhibited strong fluorescence emission in the wavelength range of 850-1200 nm. Compared with 4STP, which only showed a single emission peak in the visible light region, 4SBZ showed a shoulder peak at 905 nm. This feature may be related to the enhanced intramolecular charge transfer process in 4SBZ. Figure 10 b). Furthermore, the maximum emission peak of 6SBZ is located at 923 nm, compared to 6STP (λ). em A slight redshift occurs at 917 nm, consistent with theoretical calculations predicting a narrower bandgap for benzene ring-terminated molecules. Most importantly, the fluorescence intensities of the benzene ring-terminated molecules 4SBZ and 6SBZ in the NIR-II region are 1.6 and 1.3 times higher, respectively, than their thiophene-terminated counterparts 4STP and 6STP, demonstrating a significantly enhanced fluorescence effect. Figure 10c). The above data indicate that the introduction of terminal benzene rings can effectively enhance the NIR-II region fluorescence performance of the molecule.

[0066] 4. Study on the photophysical properties of nanoparticles Based on the significant effectiveness of the aforementioned terminal benzene ring cyclization strategy in enhancing NIR-II fluorescence performance at the molecular level, this invention further extends it to nanomaterial systems to explore the photophysical behavior of such molecules in the aggregated state, and systematically evaluates whether the introduction of terminal benzene rings can achieve synergistic optimization of fluorescence intensity at the nanoscale. Four types of nanoparticles—4SBZ-F NPs, 4STP-F NPs, 6SBZ-F NPs, and 6STP-F NPs—were constructed using a nanoprecipitation method with a hydrophobic core composed of small molecules and coated with the amphiphilic polymer F127. Figure 11 ).

[0067] First, the surface morphology and hydration size of the material were characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). Figure 12 The results showed that all four nanomaterials exhibited good monodisperse spherical morphology, but their hydration diameters differed to some extent. Specifically, the sizes of the benzene ring-based nanoparticles 4SBZ-F NPs and 6SBZ-F NPs were 73.92 nm and 68.63 nm, respectively. Figure 12 a, Figure 12 c), both are smaller than the corresponding thiophene nanoparticles 4STP-F NPs (157.3 nm) and 6STP-F NPs (92.97 nm). Figure 12 b, Figure 12 d). This indicates that the introduction of benzene ring-terminated molecules facilitates the formation of smaller, more concentrated nanoparticles. This may be due to the higher planarity of the benzene ring-terminated molecules, which promotes more ordered molecular stacking during the self-assembly of nanoprecipitates and ultimately forms smaller nanoparticles. Furthermore, the sizes of all four nanomaterials conform to the optimal size range (40-200 nm) for passive targeting of solid tumors through the EPR effect, which is beneficial for avoiding liver retention and enhancing tumor tissue penetration.

[0068] To systematically investigate the regulatory effect of terminal benzene ring cyclization on the fluorescence properties of nanomaterials, the absorption and fluorescence spectra of aqueous solutions of 4SBZ-F NPs, 4STP-F NPs, 6SBZ-F NPs, and 6STP-F NPs were measured at the same absorbance (0.1), with ICG in DMSO solution as a reference (λ). abs = 715 nm, λ em = 810 nm, Φ in dimethyl sulfoxide ICG Calculate the fluorescence quantum yield of each material (e.g., = 13%). Figure 13As shown in Figure a, for 6SBZ-F NPs and 6STP-F NPs with the same donor structure, they have similar absorption bands in the 600-900 nm range, with the maximum absorption peak (λ) being the same. abs The absorption peaks of the other two groups of 4SBZ-F NPs and 4STP-F NPs, respectively, were at 694 nm and 714 nm; the absorption peaks of the other two groups, 4SBZ-F NPs and 4STP-F NPs, appeared at 675 nm and 712 nm, respectively, within the 600-900 nm range. Under 808 nm laser irradiation, solutions with the same absorbance (0.1) exhibited bright NIR-II emission extending to 1200 nm. Figure 13 b). Thiophene-based and benzene-based molecules with the same donor structure exhibit similar fluorescence spectral characteristics: among them, the maximum emission peaks (λ) of 4SBZ-F NPs and 4STP-F NPs are similar. em The maximum emission peaks of 4SBZ-F NPs and 6STP-F NPs are located at 926 nm and 936 nm, respectively, with a shoulder peak at 220 nm; the maximum emission peaks of 6SBZ-F NPs and 6STP-F NPs are located at 880 nm and 890 nm, respectively, with a shoulder peak at 180 nm. In contrast, the non-benzene-ring-terminated nanoparticles exhibit redshifted absorption and emission, which may be due to the formation of aggregated structures favorable for redshift within the nanoparticles through specific molecular stacking (such as J-aggregation). Most importantly, compared with 4STP-F NPs, 6STP-F NPs show significantly enhanced fluorescence emission (4SBZ-F NPs vs. 6SBZ-F NPs). Figure 13 b). Using commercial dye ICG as a reference (QY: 13% in DMSO), QY 6SBZ-F (0.88%) can reach QY 6STP-F (0.72%) is 1.22 times that of QY 4SBZ-F (2.51%) compared to QY 4STP-F (1.46%) reached a surprising 1.70 times ( Figure 13 c). These data are highly consistent with the expectation of leveraging the unique properties of terminal benzene rings to enhance the fluorescence intensity of nanomaterials, strongly confirming the effectiveness of the terminal benzene ringization strategy in improving the fluorescence performance of nanomaterials. This strategy not only performs well in dilute solutions (single-molecule state) but also exhibits a significant fluorescence enhancement effect in aggregated states (nanostructures), ultimately achieving synergistic optimization from the molecular to the nanoscale. In particular, 4SBZ-F NPs possess a high fluorescence quantum yield of up to 2.51% in water, and this highly efficient near-infrared II emission characteristic makes it show great application potential in in vivo imaging scenarios.

[0069] For the detailed process and data of calculating the fluorescence quantum yield of each material, please refer to [link / reference]. Figure 14 See Table 3. Figure 14 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 4SBZ-F NPs aqueous solution. (b) Corresponding fluorescence spectrum of 4SBZ-F NPs. (c) Absorption spectrum of 4STP-F NPs aqueous solution. (d) Corresponding fluorescence spectrum of 4SBZ-F NPs. (e) Absorption spectrum of 6SBZ-F NPs aqueous solution. (f) Corresponding fluorescence spectrum of 6SBZ-F NPs. (g) Absorption spectrum of 6STP-F NPs aqueous solution. (h) Corresponding fluorescence spectrum of 6STP-F NPs. (i) Curves showing the integral fluorescence intensity of the four nanoparticles under 808 nm excitation as a function of absorbance. 850 nm, (j) Absorption spectrum of ICG in DMSO solution at 1200 nm. (k) Corresponding fluorescence spectrum of ICG. (f) Curve of integral fluorescence intensity of ICG in DMSO under 808 nm excitation as a function of absorbance. 850 nm, (1200 nm). The absorption spectra of five solutions with absorbances at 808 nm of 0.02, 0.04, 0.06, 0.08, and 0.10 were measured, respectively: aqueous solutions of 4SBZ-F NPs, 4STP-F NPs, 6SBZ-F NPs, 6STP-F NPs, and ICG in dimethyl sulfoxide solution. Their respective fluorescence spectra under 808 nm excitation were compared, and the slope was obtained by plotting an integrated fluorescence spectrum-absorbance fitting curve. According to the following formula:

[0070] Calculate the fluorescence quantum yield for each solvent. Using the ICG fluorescence quantum yield in DMSO (Φ = 13%) as a reference, calculate the refractive index η for different solvents. H2O = 1.33, η DMSO = 1.479.

[0071] The parameters are recorded in Table 3. The fluorescence quantum yield Φ is calculated using the relative method. 4SBZ-F NPs = 2.51%, Φ 4STP-F NPs = 1.46%, Φ 6SBZ-F NPs = 0.88%, Φ 6STP-F NPs = 0.72%, indicating that the terminal benzene cyclization strategy can effectively enhance the fluorescence brightness of near-infrared II nanomaterials.

[0072] Table 3 Calculation of fluorescence quantum yield in water

[0073] To further investigate the dominant factors and underlying mechanisms of the difference in fluorescence performance between benzene ring-terminated and non-benzene ring-terminated molecules, the photothermal properties of aqueous solutions of 4SBZ-F NPs, 4STP-F NPs, 6SBZ-F NPs, and 6STP-F NPs at concentrations of 25 μg / mL were measured using a handheld thermophotometer. For the same volume (500 μL) of solution, at (808 nm, 1 W / cm²)... 2 Under laser irradiation, thiophene-based nanoparticles 4STP-F NPs and 6STP-F NPs rapidly increased in temperature from 28.1℃ to 56.5℃ and 59.2℃ respectively within 15 minutes, while benzene ring-based nanoparticles 4SBZ-F NPs and 6SBZ-F NPs only increased in temperature from room temperature to 53.3℃ and 54.1℃ respectively. Figure 16 ). Calculate the photothermal conversion efficiency (PCE) of different nanoparticles. For example... Figure 15 , Figure 16 As shown in Table 4, the photothermal conversion efficiencies of the benzene ring-terminated nanomaterials 4SBZ-F NPs and 6SBZ-F NPs were 21.4% and 36.2%, respectively, both showing a decrease compared to the corresponding non-benzene ring-terminated nanomaterials 4STP-F NPs (PCE = 22.1%) and 6SBZ-F NPs (PCE = 44.5%). This result confirms that the introduction of benzene ring end groups can effectively suppress the nonradiative relaxation process of molecules, and corroborates the conclusion in the aforementioned theoretical calculations that the higher coplanarity of benzene ring molecules restricts intramolecular vibrations and rotations. Together, they reveal the intrinsic mechanism by which the "terminated benzene ringization strategy" enhances the fluorescence performance of materials: by enhancing molecular rigidity and planarity, regulating the excited-state energy relaxation path to promote radiative transitions, and ultimately achieving a significant enhancement of fluorescence performance.

[0074] Table 4. Calculation of photothermal conversion efficiency for four types of nanoparticles

[0075] 5. Characterization of the optimal in vitro properties of nanoparticles Based on the excellent fluorescence performance (Φ = 2.51%) exhibited by 4SBZ-F NPs in the NIR-II region, they were selected as candidate materials for subsequent application research. Subsequently, to verify the application advantages of 4SBZ-F NPs, the NIR-II imaging performance of the commercial dye indocyanine green (ICG), IR-1061, and 4SBZ-F NPs was compared. Figure 17Considering the hydrophobicity of IR-1061, a DCM solution of IR-1061 was prepared for testing. For the same concentration (25 μg / mL) and the same volume (1 mL) of 1CG aqueous solution, the fluorescence intensity of IR-1061 DCM solution and 4SBZ-F NPs aqueous solution under 808 nm laser light was 4.32 times that of dye IR-1061 and 14.5 times that of ICG. Figure 17 a). Constant illumination (808 nm, 1 W / cm²) 2 After 30 minutes, 4SBZ-F NPs still maintained more than 85% of their initial brightness, while ICG suffered severe photodegradation, with its fluorescence intensity rapidly decreasing to 10% of its initial brightness within 17 minutes. Figure 17 (b) The above results demonstrate that 4SBZ-F NPs not only exhibit significantly superior NIR-II fluorescence brightness compared to the two commercial dyes, but also demonstrate excellent photostability: a 14.5-fold increase in fluorescence intensity and far greater photobleaching resistance than ICG give them a clear advantage in applications requiring long-duration, high signal-to-noise ratio imaging. Compared to IR-1061, which requires solubility in organic solvents, 4SBZ-F NPs still achieve a 4.32-fold increase in brightness in aqueous solution, further highlighting their comprehensive application potential as a water-soluble, high-brightness, and highly stable NIR-II imaging nanomaterial.

[0076] To assess the cytotoxicity of the materials, the MTT assay was used to determine the cytotoxicity of materials treated with different concentrations of 6TCl-PS. 200 Cell viability of mouse embryonic osteoblasts MC3T3 after NPs incubation ( Figure 18 The results showed that cell viability remained above 80% within a concentration range of 20-100 μg / mL. Even at the highest concentration (100 μg / mL), MC3T3 cell viability still reached 84%, showing no significant decrease compared to the control group without nanomaterial treatment. These data indicate that 4SBZ-F NPs did not significantly inhibit normal cell metabolism within the measured concentration range, exhibiting low cytotoxicity and good biocompatibility, demonstrating their excellent application value in mediating in vivo imaging.

[0077] 6. Imaging of blood vessels, lymph nodes, and tumors With their excellent photophysical properties and superior biocompatibility, 4SBZ-F NPs demonstrate promising potential for in vivo imaging applications. To further evaluate the NIR-II fluorescence imaging performance of 4SBZ-F NPs, vascular, lymph node, and tumor imaging in mice was performed sequentially using 4SBZ-F NPs.

[0078] Immediately after a tail vein injection of 200 μL (250 μg / mL) of PBS solution containing 4SBZ-F NPs, real-time imaging of the entire vascular system of mice was performed using a near-infrared II in vivo imaging system. Figure 19 As shown in Figure a, the complete circulatory system in mice can be clearly observed under 808 nm laser irradiation. To preliminarily evaluate the vascular imaging resolution of 4SBZ-F NPs, the fluorescence signal at the same cross-section in the main vascular region of the abdomen was quantitatively analyzed. The cross-section is marked by a red line. Figure 19 a). Gaussian fitting was performed on the fluorescence intensity values ​​of blood vessels in different locations ( Figure 19 (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 of the mouse vascular fluorescence signal was 0.67 mm, indicating that 4SBZ-F NPs can achieve high-resolution vascular imaging. Further quantitative analysis of the fluorescence signal between the same area of ​​the vascular region and the adjacent background region yielded a signal-to-background ratio (SBR). The image SBR reached 11.89, indicating high imaging contrast and good imaging quality. These results collectively demonstrate that 4SBZ-F NPs exhibit high imaging resolution (FWHM = 0.67 mm) and contrast (SBR = 11.89) under 808 nm excitation and 1000 nm long-pass filter acquisition conditions, providing clear vascular imaging signals and meeting the needs for deep and detailed vascular identification. This provides a potential tool for precise visualization and dynamic analysis of clinical vascular structures.

[0079] 4SBZ-F NPs in PBS solution (20 μL, 250 μg / mL) were subcutaneously injected into the right hind paw of mice. Ten minutes after injection, near-infrared spectroscopy (NIRS) was used to acquire images of the hind limb lymph nodes and observe lymphatic drainage. The images showed that the popliteal lymph nodes could be identified along the afferent lymphatic vessels 10 minutes after injection. Figure 19 c-Ⅰ). Subsequently, the efferent lymphatic vessels and sacral lymph nodes were also visible ( Figure 19 (c-Ⅲ). Within 90 minutes after injection, the fluorescence signal of 4SBZ-F NPs in the lymphatic system continued to increase; from 30 minutes onwards, bright globular lymph nodes and slender lymphatic vessels remained clearly visible. Further quantitative analysis... Figure 19 The fluorescence signal at the lymph nodes in c-VI showed a half-width at half-maximum (FWHM) of 0.47 mm and a signal-to-background ratio (SBR) of 6.67. Imaging results and analysis indicate that 4SBZ-F NPs can achieve long-term dynamic tracking of lymphatic drainage with high resolution and high contrast, and can be used to detect physiological and pathological processes related to the circulatory system in organisms, including lymphatic drainage.

[0080] 4T1 tumor-bearing mice were injected intravenously via tail vein with PBS solution containing 4SBZ-F NPs (200 μL, 250 μg / mL), and NIR-II fluorescence imaging was subsequently performed on the mice at different time points. Figure 20 a). Benefiting from the enhanced permeability and retention (EPR) effect, 4SBZ-F NPs continuously accumulate at the tumor site, and the fluorescence signal in the tumor area gradually increases over time, reaching a peak at 44 h post-injection. Figure 20 b). The tumor outline remained clearly visible throughout the 4SBZ-F NPs-mediated NIR-II fluorescence imaging process, with the 44th hour post-injection serving as the optimal time point for guiding surgical resection. 116 hours post-injection, in vitro imaging of mouse tumor tissue and major organs (heart, liver, spleen, lungs, kidneys) was performed. Figure 20 c, Figure 20 d). Imaging and quantitative results showed that strong fluorescence signals were observed only in tumor tissue and the liver, while the fluorescence signals in other organs were weaker, indicating that 4SBZ-F NPs could be well enriched at the tumor site and could be partially metabolized by the liver after intravenous injection. These results demonstrate that 4SBZ-F NPs are efficiently enriched at the tumor site through a passive targeting mechanism, exhibit excellent fluorescence imaging contrast within the NIR-II window, and can clearly display tumor boundaries, demonstrating significant clinical application value in the fields of precise tumor imaging and surgical navigation.

[0081] 7. Biosafety assessment To verify the biosafety of 4SBZ-F NPs, the major organs of the treated mice were subjected to H&E staining and histopathological evaluation. Figure 21 As shown, compared with the control group which only received PBS buffer, the tissue structures of key organs such as the heart, liver, spleen, lungs, and kidneys remained intact in the experimental group, with no obvious inflammatory cell infiltration, nor any abnormal pathological changes such as tissue degeneration or necrosis. These results confirm that 4SBZ-F NPs exhibit good biocompatibility in vivo, without causing detectable tissue toxicity, meeting the basic biosafety requirements for further clinical translation.

[0082] In summary, this invention proposes for the first time a molecular design strategy of "terminal benzene ringization" for the optimization of the brightness of NIR-II small molecules, and based on this, a series of small molecules 4SBZ, 4STP, 6SBZ, and 6STP with benzene rings and thiophene as terminal groups were designed and synthesized.

[0083] (1) The results of molecular structure optimization and fluorescence performance characterization show that the introduction of benzene ring end groups can enhance molecular planarity. The fluorescence intensity of benzene ring small molecules (4SBZ, 6SBZ) in dilute solutions is increased by 1.6 times and 1.3 times respectively compared with the corresponding thiophene end group molecules, which verifies the effectiveness of the strategy.

[0084] (2) Further investigation was conducted on the photophysical behavior of small molecules with different end groups in the aggregated state within the nanostructure. Benzene ring-based nanoparticles (4SBZ-F NPs, 6SBZ-F NPs) still exhibited superior fluorescence performance, indicating that the introduction of end-group benzene rings can effectively achieve synergistic optimization at the molecular and nanoscale levels. Subsequently, photothermal response experiments revealed the intrinsic mechanism of the improved fluorescence performance: the introduction of benzene ring end groups can effectively suppress the nonradiative relaxation process of molecules, regulate the excited-state energy relaxation path to promote radiative transitions, and ultimately achieve a significant enhancement in fluorescence performance.

[0085] (3) Material 4SBZ-F NPs exhibits excellent NIR-II fluorescence brightness (Φ = 2.51%), good biocompatibility, and significantly improved photostability compared to the commercial dye ICG. It can be efficiently enriched at tumor sites through enhanced penetration and retention (EPR) effects. Under low-power laser (1 W / cm²), 2 With a short exposure time (30 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.

[0086] The "terminal benzene ring cyclization" strategy proposed in this invention provides a new and effective molecular design method for developing high-brightness, highly biocompatible NIR-II organic nanomaterials. The 4SBZ-F NPs constructed based on this strategy have shown clear translational potential in clinical applications such as deep tissue imaging, early tumor detection, and intraoperative navigation.

[0087] 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.

[0088] 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 nanoparticle, characterized in that, The organic nanoparticles are 4SBZ-F NPs, 6SBZ-F NPs, 4STP-F NPs, or 6STP-F NPs.

2. The method for preparing organic nanoparticles as described in claim 1, characterized in that, The preparation method includes the following steps: S1, Dissolve the solid organic molecule in tetrahydrofuran to obtain a molecular stock solution, wherein the organic molecule is 4SBZ, 4STP, 6SBZ or 6STP; S2, F127 powder is dissolved in tetrahydrofuran to obtain a coating agent stock solution; S3. The molecular stock solution and the coating agent stock solution are mixed evenly, then injected into pure water, ultrasonically treated, and then tetrahydrofuran is removed. After filtration, organic nanoparticles are obtained.

3. The method for preparing organic nanoparticles according to claim 2, characterized in that, The organic molecule was prepared by Knoevenagel condensation reaction.

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

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

6. The method for preparing organic nanoparticles according to claim 2, characterized in that, When the molecular stock solution and the coating agent stock solution are mixed, the mass ratio of organic molecules to coating agent is 1:

20.

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

9.

8. The application of organic nanoparticles, characterized in that, The organic nanoparticles of claim 1 are used for bioimaging.

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