Nanoparticle with fluorescence enhancement performance and preparation method and application thereof

Abstract

This invention discloses a fluorescence-enhancing nanoparticle, its preparation method, and its applications, relating to the fields of organic fluorescent molecules and bioimaging technology. The nanoparticle comprises: NIR-II fluorescent molecules and an amphiphilic polymer encapsulating the NIR-II fluorescent molecules; the amphiphilic polymer has a hydrophilic segment and a rigid hydrophobic segment. The nanoparticle of this invention, based on "carrier microenvironment engineering," utilizes the rigid confinement and interfacial isolation provided by the amphiphilic polymer with the rigid hydrophobic segment for dual brightening, serving as a universal brightening platform applicable to various NIR-II fluorescent molecules. This nanoparticle can significantly improve aqueous phase quantum yield, enhance contrast and resolution in in vivo vascular imaging, improve deep tissue penetration, increase imaging sensitivity, and facilitate detection under low signal conditions. It also exhibits good stability and biocompatibility, supporting lesion imaging and intraoperative navigation applications.

Description

Technical Field

[0001] This invention relates to the fields of organic fluorescent molecules and bioimaging technology, and in particular to a nanoparticle with fluorescence enhancement properties, its preparation method, and its application. Background Technology

[0002] Near-infrared II (NIR-II, typically referring to wavelengths greater than 1000 nm) fluorescence imaging technology, due to its lower tissue scattering, stronger photon penetration, and weaker tissue autofluorescence background, can obtain higher contrast and clearer structural information in scenarios such as vascular imaging, lesion visualization, and intraoperative navigation. With the increasing demand in clinical and research fields for "deep, microstructures, and real-time navigation," NIR-II fluorescence imaging places higher demands on probe brightness, signal-to-noise ratio (SBR), and spatial resolution.

[0003] However, most existing organic NIR-II phosphors have low quantum yield (QY) in actual aqueous environments or in vivo imaging conditions, resulting in insufficient emission brightness and limited background suppression capabilities. This limits the imaging SBR and spatial resolution, making it difficult to meet the "bright and stable" requirements of high-quality fine imaging and surgical navigation applications.

[0004] Low QY values ​​in existing organic NIR-II phosphors are usually caused by a combination of factors, including but not limited to: (1) Enhanced nonradiative loss related to band gap: In order to obtain long-wavelength emission, molecular design often reduces the band gap by enhancing the DA effect and extending conjugation; however, reducing the band gap will enhance molecular vibrational coupling and increase the probability of nonradiative decay, making it difficult to maintain QY at a high level.

[0005] (2) Quenching in the water environment: Water molecules have a high-frequency OH vibration mode, and the strong polar environment of the water phase is easy to couple with the excited state, thereby inducing energy to dissipate through non-radiative channels; therefore, the QY of the phosphor in the water phase or in the body environment often decreases further.

[0006] (3) Non-radiative loss in aggregated state: Organic NIR-II dyes mostly work in the bulk in a nano-aggregate state. After aggregation, factors such as intramolecular rotational / vibrational degrees of freedom, stacking defects and microenvironment instability can easily introduce additional non-radiative channels, further reducing luminescence efficiency.

[0007] The above problems indicate that optimization at the molecular level alone is often insufficient, and synergistic regulation at multiple scales, including "molecule-aggregate-nanosystem-microenvironment / interface", is necessary.

[0008] To improve the luminescence and imaging performance of NIR-II, existing technologies mainly adopt the following approaches: (1) Molecular structure design approach: Redshift and enhanced aggregated state luminescence can be achieved by strengthening the DA structure, extending conjugation, introducing steric hindrance or twisting to suppress excessive π-π stacking. This approach can improve the photophysical properties of materials to a certain extent, but it is usually difficult to overcome the inherent contradiction of "long-wavelength emission leading to non-radiative enhancement" and the external constraint of quenching in the water environment at the same time.

[0009] (2) Nano-encapsulation route: Amphiphilic carriers such as F127, PCL-PEG, PLGA-PEG, and DSPE-PEG are used to encapsulate hydrophobic dyes in nano-sized form to improve water dispersibility and biocompatibility. This route has mature technology and strong operability, but most carriers mainly provide general hydrophobic cavities and have limited contribution to conformational rigidity and interface isolation, so the improvement of QY and imaging quality is often insufficient.

[0010] (3) Inorganic / crosslinked shell isolation route: Water molecule contact is reduced by using a silicon shell or crosslinked polymer shell, thereby reducing water-induced quenching. This route can improve stability to a certain extent, but it usually has problems such as complex preparation process, insufficient structural controllability and degradability, and increased metabolic burden in vivo, which affect further transformation and application.

[0011] Although the above-mentioned technical approaches can improve the usability of NIR-II imaging to some extent, they still generally suffer from the following drawbacks, which constitute the most relevant technical pain points to the application needs in this field: (1) "Can encapsulate but insufficient confinement and isolation": Conventional amphiphilic carriers mostly provide only a general hydrophobic microenvironment, which is difficult to achieve strong confinement and conformational stiffening of phosphors, resulting in the inability to effectively suppress non-radiative decay; at the same time, it is difficult to form a sufficiently dense hydrophobic barrier at the carrier / nanoparticle interface, and vibrational coupling quenching of interfacial water still exists significantly. Therefore, under the same dye and conditions, the imaging SBR and resolution improvement of conventional carrier systems are limited, and in applications such as vascular imaging, they often exhibit problems such as low SBR and wide FWHM.

[0012] (2) Insufficient margin for deep imaging: In the test of tissue penetration or deep imaging, the signal of conventional encapsulation system decays rapidly with the increase of tissue thickness, and is easily close to the background and difficult to distinguish; when the tissue thickness is large, the control system may show a signal that is close to unrecognizable, while the enhancement system can still maintain a significant difference from the background, indicating that the existing conventional encapsulation scheme is insufficient to guarantee deep penetration.

[0013] (3) Insufficient universality and reproducibility: Many brightening strategies are effective for specific molecules, but their performance is inconsistent for NIR-II phosphors with different structural types, making it difficult to form a stable and reproducible universal platform. A comparison of different carrier systems shows that some carriers "cannot improve fluorescence efficiency at all", while functionalized carriers with stronger hydrophobic rigid segments and the ability to achieve "strong confinement of aggregates + effective isolation of interfaces" are more likely to show a consistent trend of QY / emission intensity improvement in multiple dye systems.

[0014] In summary, there is an urgent need in this field for a carrier / encapsulation solution that can simultaneously achieve strong confinement rigidity and hydrophobic isolation at the nanoscale, in order to significantly improve the quantum yield and imaging SBR of NIR-II organic phosphors in aqueous and in vivo environments, while taking into account deep imaging capabilities and universality across molecular systems, thereby better meeting the needs of high-quality in vivo fine imaging and navigation applications. Summary of the Invention

[0015] In view of the shortcomings of the prior art, the purpose of this invention is to provide a nanoparticle with fluorescence enhancement properties, its preparation method and application, aiming to solve the problem that the quantum yield of existing NIR-II phosphors is low under actual aqueous environment or in vivo imaging conditions, which leads to limitations in probe brightness, signal-to-noise ratio and spatial resolution.

[0016] The technical solution of the present invention is as follows: In a first aspect, a nanoparticle with fluorescence enhancement properties is provided, comprising: an NIR-II fluorescent molecule and an amphiphilic polymer encapsulating the NIR-II fluorescent molecule; The amphiphilic polymer has a hydrophilic segment and a rigid hydrophobic segment.

[0017] In a preferred embodiment, the hydrophilic segment is selected from at least one of polyethylene glycol, methoxy polyethylene glycol, poly(N,N-dimethylacrylamide), polymethyl methacrylate oligoethylene glycol, polyglycerol monoacrylate, and polyacrylamide, or a deuterated form thereof.

[0018] In a preferred embodiment, the rigid hydrophobic segment is selected from at least one deuterated product of polystyrene, polyhydroxypropyl methacrylate, polyoctadecyl methacrylate, polyvinyl biphenyl, poly[1-(4-vinylphenyl)-1,2,2-triphenyl]ethylene, polybenzyl methacrylate, polyvinylnaphthalene, polyvinylpyrene, poly4-bromostyrene, and polypentafluorostyrene.

[0019] In a preferred embodiment, the amphiphilic polymer is selected from at least one of the following structures: ; In the structural formula, n and m represent the degree of aggregation, where n is an integer between 26 and 222, and m is an integer between 15 and 1260. b This is the block segment symbol in the amphiphilic polymer.

[0020] In a preferred embodiment, the amphiphilic polymer is selected from at least one of the following structures: ; In the structural formula, n represents the degree of aggregation, which is an integer between 51 and 144. b This is the block segment symbol in the amphiphilic polymer.

[0021] In a preferred embodiment, the NIR-II fluorescent molecule is selected from at least one of the following structures: ; R1 and R6 are independently selected from substituted or unsubstituted aryl, substituted or unsubstituted triphenylamino, substituted or unsubstituted tristyryl, and substituted or unsubstituted tetrastyryl. R2 and R5 are independently selected from hydrogen, halogen, C1-C30 straight-chain or branched alkyl, C1-C30 straight-chain or branched alkoxy, C3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted triphenylamine, substituted or unsubstituted tristyryl, substituted or unsubstituted tetrastyryl. R3 and R4 are independently selected from halogens, C1-C30 straight-chain or branched alkyl groups, C1-C30 straight-chain or branched alkoxy groups, C3-C6 cycloalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted triphenylamine groups, substituted or unsubstituted tristyryl groups, and substituted or unsubstituted tetrastyryl groups.

[0022] In a preferred embodiment, the NIR-II fluorescent molecule is selected from at least one of the following structures: .

[0023] Secondly, a method for preparing the nanoparticles described in the first aspect is provided, comprising the steps of: The NIR-II fluorescent molecule and the amphiphilic polymer were dissolved in an organic solvent to obtain a mixed solution; The mixed solution was added dropwise to water under ultrasonic conditions to obtain nanoparticles.

[0024] In a preferred embodiment, the organic solvent is selected from at least one of tetrahydrofuran, dimethyl sulfoxide, and dichloromethane.

[0025] Thirdly, the application of nanoparticles in fluorescence imaging as described in the first aspect is provided.

[0026] By adopting the above technical solution, the present invention has at least the following beneficial effects: (1) Significantly improves the quantum yield of aqueous phase: Compared with conventional carrier encapsulation systems, this invention uses an amphiphilic polymer with a rigid hydrophobic segment as a carrier for encapsulation, which can limit the improvement of the fluorescence quantum yield of NIR-II fluorescent molecules. Moreover, it has a significant improvement effect on fluorescent molecules with different structures.

[0027] (2) Improved contrast and resolution of in vivo vascular imaging: Under the same molecular and drug administration conditions, the nanoparticles of the present invention can achieve higher SBR and narrower FWHM; and exhibit better resolution and contrast in different vascular locations.

[0028] (3) Enhanced deep tissue penetration capability: In the ex vivo tissue (chicken breast) penetration model, when the thickness reaches 34 mm, the signal of the control nanoparticles is almost indistinguishable from the background, while the nanoparticles of the present invention can still maintain a signal intensity more than twice that of the background, thus providing a higher margin for deep imaging.

[0029] (4) Improved imaging sensitivity and facilitated detection under low signal conditions: In in vitro concentration gradient imaging, the nanoparticles of the present invention maintain a stronger signal over a wider concentration range and can still achieve reliable detection at low concentrations under low power excitation, demonstrating their advantages in brightness and sensitivity.

[0030] (5) Good stability and biocompatibility: The nanoparticles of the present invention can maintain colloidal stability for a long time in water and serum-containing buffer systems and exhibit good photostability; at the same time, no obvious pathological abnormalities were found in the major organs H&E, and the hematological and serum biochemical indicators were within the normal reference range, indicating good in vivo biocompatibility.

[0031] (6) Support for lesion imaging and intraoperative navigation: For example, a signal peak can be observed about 72 hours after drug administration in the relevant model, and smaller nodules (average diameter about 2.08±0.52 mm) can be identified and removed under fluorescence guidance, demonstrating the application potential of the nanoparticles of the present invention in imaging navigation. Attached Figure Description

[0032] Figure 1 It is mPEG 2000 - b -PS 1 H nuclear magnetic resonance spectrum.

[0033] Figure 2 It is mPEG 2000 - b - SEC map of PS.

[0034] Figure 3 It is POEGMA n - b -PS mof 1 H nuclear magnetic resonance spectrum.

[0035] Figure 4 It is POEGMA n - b -PS m SEC map.

[0036] Figure 5 It is PDMA n - b -PS m of 1 H nuclear magnetic resonance spectrum.

[0037] Figure 6 It is PDMA n - b -PS m SEC map.

[0038] Figure 7 It is TTT6,2-B. 1 H nuclear magnetic resonance spectrum.

[0039] Figure 8 It is TTT6,2-B. 13 C nuclear magnetic resonance spectrum.

[0040] Figure 9 This is the MALDI-TOF MS spectrum of TTT6,2-B.

[0041] Figure 10 The absorption and emission wavelength spectra of TTT6,2-B are shown; the absorption peak is at 278 nm and the emission peak is at 1010 nm.

[0042] Figure 11 TTT6,2-B are respectively in mPEG- b -PS and mPEG- b - Fluorescence quantum yield results under DSPE loading; with IR-26 as the reference standard (QY = 0.05%).

[0043] Figure 12 It is TTT6,2-B in mPEG- b - Quantum yield results of nanoparticles prepared under PS encapsulation in different solvents.

[0044] Figure 13 TTT6,2-B are respectively in mPEG- b Fluorescence spectra of nanoparticles prepared by loading with PS and other control amphiphilic carriers.

[0045] Figure 14 TTT6,2-B are respectively in mPEG-b -Quantum yield calculation results of nanoparticles prepared under PS and other control amphiphilic carriers.

[0046] Figure 15 TTT6,2-B, 4TPE-4N, TBBSD, 4TPA-TQ, TTBI, and TPABT-TQ are respectively in mPEG- b -Statistical results of quantum yield of nanoparticles prepared under PS and other control amphiphilic carriers.

[0047] Figure 16 This is a graph showing the quantum yield statistics of nanoparticles prepared by TTT6,2-B under the encapsulation of other amphiphilic polymers with rigid hydrophobic segments.

[0048] Figure 17 TTT6,2-B are respectively in mPEG- b -PS and mPEG- b -Statistical results of nanoparticle size and transmission electron microscopy images of nanoparticles prepared under DSPE loading.

[0049] Figure 18 It is TTT6,2-B in mPEG- b - Figure showing the absorption and particle size storage stability of nanoparticles prepared under PS encapsulation; the solvent conditions were water and PBS solution containing 10% serum protein, and the storage period was 15 days.

[0050] Figure 19 It is TTT6,2-B in mPEG- b -Graph showing the trend of absorption and emission intensity changes of nanoparticles prepared under PS loading and the control material ICG under continuous irradiation by an 808 nm laser.

[0051] Figure 20 TTT6,2-B are respectively in mPEG- b -PS and mPEG- b -NIR-II fluorescence imaging of nanoparticles prepared under DSPE encapsulation at different concentrations in aqueous solution, and TTT6,2-B at corresponding concentrations in mPEG- b - Fluorescence imaging of nanoparticles encapsulated in PS in deuterium water.

[0052] Figure 21 TTT6,2-B are respectively in mPEG- b -PS and mPEG- b - NIR-II fluorescence imaging of nanoparticles prepared under DSPE encapsulation under conditions of covering chicken breast of different thicknesses.

[0053] Figure 22 TTT6,2-B are respectively in mPEG- b -PS and mPEG- b -NIR-II fluorescence imaging and statistical results of signal-to-noise ratio and full width at half maximum (FWHM) of nanoparticles prepared under DSPE encapsulation after administration of the same dose via tail vein.

[0054] Figure 23 TTT6,2-B are respectively in mPEG- b -PS and mPEG- b -NIR-II fluorescence imaging of subcutaneous tumors at different time points after tail vein administration of nanoparticles prepared under DSPE encapsulation with the same dose.

[0055] Figure 24 TTT6,2-B are respectively in mPEG- b -PS and mPEG- b -Statistical results of NIR-II fluorescence intensity of subcutaneous tumors at different time points after tail vein administration of nanoparticles prepared under DSPE encapsulation with the same dose.

[0056] Figure 25 It is TTT6,2-B in mPEG- b -NIR-II fluorescence imaging and fluorescence intensity statistics of nanoparticles prepared under PS encapsulation on tumors in aqueous solution and deuterated water.

[0057] Figure 26 It is TTT6,2-B in mPEG- b - A fluorescence-guided surgical navigation result of nanoparticles prepared under PS encapsulation after drug administration in a colorectal model.

[0058] Figure 27 It is TTT6,2 The theoretical calculation results of B are shown in the figure; where A is TTT6,2. The radial distribution function (RDF) of the S atom on the TBB unit in B relative to the oxygen atoms (O) in the surrounding H2O molecule; B is the mean square displacement (MSD) of the S atom on the BBTD unit in TTT6,2-B relative to the oxygen atoms (O) in the surrounding H2O molecule. Detailed Implementation

[0059] This invention provides nanoparticles with fluorescence enhancement properties, their preparation method, and applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below.

[0060] Existing near-infrared II (NIR-II) organic phosphors generally suffer from low quantum yields in aqueous and in vivo applications, limiting the signal-to-noise ratio (SBR), spatial resolution, and deep penetration capability of in vivo imaging. The main reasons for these problems include, but are not limited to: (1) Long-wave emission and bandgap-related nonradiative decay: To achieve NIR-II long-wave emission, it is usually necessary to reduce the molecular bandgap. However, a reduction in the bandgap will enhance vibrational coupling and promote nonradiative decay, resulting in a significant decrease in quantum yield.

[0061] (2) Energy loss caused by intramolecular motion and stacking defects in aggregated molecules: Organic NIR-II phosphors mostly exist in aggregate / nano states in vivo. If the confinement of the aggregates is insufficient, the intramolecular vibrational and rotational degrees of freedom and stacking defects will open additional non-radiative channels, thereby further reducing the luminescence efficiency. This indicates that relying solely on molecular-level optimization is insufficient to completely solve the problem.

[0062] (3) Interfacial water-induced quenching: The high-frequency OH vibration of water molecules in the physiological aqueous phase is easily coupled with the excited state and induces nonradiative relaxation, which further reduces the quantum yield in the aqueous / in vivo environment.

[0063] Therefore, the present invention aims to provide a nanoparticle with fluorescence enhancement properties. Without relying on complex inorganic shells or highly burdened cross-linked shells, the present invention significantly improves the aqueous quantum yield of the coated NIR-II fluorescent molecules by engineering and controlling the nanoscale microenvironment of the fluorophore, while suppressing non-radiative loss of aggregated state and reducing water-induced quenching at the interface, thereby further improving the SBR, resolution and deep penetration capability of in vivo imaging.

[0064] This invention provides a nanoparticle with enhanced fluorescence properties, comprising: NIR-II fluorescent molecules and an amphiphilic polymer encapsulating the NIR-II fluorescent molecules; The amphiphilic polymer has a hydrophilic segment and a rigid hydrophobic segment.

[0065] Specifically, to achieve the above objectives, the present invention provides nanoparticles with fluorescence enhancement properties, as detailed below: (1) Carrier selection and self-assembly structure A self-assembled structure (nanoparticle) is formed using an amphiphilic polymer with a strong hydrophobic structure, and NIR-II fluorescent molecules are encapsulated in the hydrophobic phase / hydrophobic core of the nanoparticle. The hydrophobic segments of the amphiphilic polymer simultaneously possess high chain rigidity or a high glass transition temperature (Tg) to construct a more "rigid" hydrophobic microenvironment.

[0066] (2) The dual brightening mechanism of "rigid confinement + interface isolation" Unlike conventional carriers that only provide a general hydrophobic cavity, the amphiphilic polymer of this invention provides the following dual functions for NIR-II fluorescent molecules: 1) Rigid confinement: The rigid hydrophobic segment interacts more strongly with NIR-II fluorescent molecules and promotes more compact co-assembly and stacking, thereby restricting the vibrational / rotational degrees of freedom within the molecule, reducing vibrational coupling and suppressing nonradiative transitions, and improving quantum yield. Furthermore, this confinement effect can be further enhanced by increasing the molecular weight of the rigid hydrophobic segment or optimizing the polymer ratio (for example, changing the polymer ratio can show a gradual increase in quantum yield and there is an optimal ratio).

[0067] 2) Interfacial isolation: The strong hydrophobicity of the rigid hydrophobic segment drives the formation of a denser hydrophobic shell / interface layer, reducing the entry of water molecules into the nanoparticle interface and reducing water-induced quenching caused by OH vibrational coupling.

[0068] (3) Verification method of interface vibration coupling To verify the role of interfacial water vibrational coupling in quenching, the nanoparticle dispersion medium can be replaced with D2O instead of H2O. Since the OD vibrational coupling is weaker, the quantum yield can be further improved, thus corroborating the contribution of "interfacial isolation / external matrix regulation" to brightening.

[0069] (4) Platformization and universality The solution of this invention is based on "carrier microenvironment engineering" and does not rely on customized modification of a single dye structure. Through synergistic regulation at the aggregate level and the aggregate-external matrix interface level, it is beneficial to form a universal brightening platform applicable to NIR-II phosphors with diverse structures.

[0070] In some embodiments, the hydrophilic segment of the amphiphilic polymer is selected from at least one of polyethylene glycol (PEG), methoxy polyethylene glycol (mPEG), poly(N,N-dimethylacrylamide) (PDMA), poly(poly(methacrylic acid) oligoethylene glycol) (POEGMA), poly(PGMAc) and polyacrylamide (PAAm), or a deuterated thereof.

[0071] In some embodiments, the rigid hydrophobic segment of the amphiphilic polymer is selected from at least one of polystyrene (PS), polyhydroxypropyl methacrylate (PHPMA), polyoctadecyl methacrylate (PSMA), polyvinyl biphenyl (PBP), poly[1-(4-vinylphenyl)-1,2,2-triphenyl]ethylene (PTPE), polybenzyl methacrylate (PBzMA), polyvinyl naphthalene (PNAP), polyvinylpyrene (PYE), poly4-bromostyrene (PSBr), and polypentafluorostyrene (PFS), and its deuterated derivatives.

[0072] In some embodiments, the amphiphilic polymer is selected from at least one of the following structures: ; In the structural formula, n and m represent the degree of aggregation, where n is an integer between 26 and 222, and m is an integer between 15 and 1260. b This is the block segment symbol in the amphiphilic polymer.

[0073] In some embodiments, the amphiphilic polymer is selected from at least one of the following structures: ; In the structural formula, n represents the degree of aggregation, which is an integer between 51 and 144. b This is the block segment symbol in the amphiphilic polymer.

[0074] In some embodiments, the NIR-II fluorescent molecule is selected from at least one of the following structures: ; R1 and R6 are independently selected from substituted or unsubstituted aryl (benzene ring and naphthyl), substituted or unsubstituted triphenylamine, substituted or unsubstituted tristyryl, and substituted or unsubstituted tetrastyryl. R2 and R5 are independently selected from hydrogen, halogen, C1-C30 straight-chain or branched alkyl, C1-C30 straight-chain or branched alkoxy, C3-C6 cycloalkyl, substituted or unsubstituted aryl (benzene ring and naphthyl), substituted or unsubstituted triphenylamino, substituted or unsubstituted tristyryl, substituted or unsubstituted tetrastyryl. R3 and R4 are independently selected from halogens, C1-C30 straight-chain or branched alkyl groups, C1-C30 straight-chain or branched alkoxy groups, C3-C6 cycloalkyl groups, substituted or unsubstituted aryl groups (benzene ring and naphthyl), substituted or unsubstituted triphenylamine, substituted or unsubstituted tristyryl, and substituted or unsubstituted tetrastyryl. In addition, π-bridges (thiophenes with or without substituents) can be independently selected as thiophene[3,2-b]thiophene, dithiophene[3,2-B:2',3'-D]thiophene, or benzo[C]thiophene.

[0075] Specifically, the synthetic route for the aforementioned NIR-II fluorescent molecules is as follows: Using 4,7-dibromobenzobisthiadiazole and substituted tributyl(thiophene-2-yl)tinane as starting materials, the target product was obtained via a Stille reaction. The synthetic route is shown below: ; The reaction route uses the following reagents and reaction conditions: tetrakis(triphenylphosphine)palladium, dry toluene, reaction temperature of 105℃, and nitrogen atmosphere.

[0076] In some embodiments, the NIR-II fluorescent molecule is selected from at least one of the following structures: .

[0077] This invention provides a method for preparing nanoparticles as described above, comprising the following steps: The NIR-II fluorescent molecule and the amphiphilic polymer were dissolved in an organic solvent to obtain a mixed solution; The mixed solution was added dropwise to water under ultrasonic conditions to obtain nanoparticles.

[0078] In some embodiments, the organic solvent is selected from at least one of tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), and dichloromethane (DCM).

[0079] The embodiments of the present invention provide the application of nanoparticles as described above in fluorescence imaging.

[0080] The present invention will be further described below through specific embodiments.

[0081] Example 1 This embodiment provides an amphiphilic polymer mPEG. n - b -PS m (or mPEG) 2000 - b -PS, or mPEG for short- b -PS), and its preparation method is as follows: I. Synthesis of mPEG with hydrophilic segment:

[0082] (1) Synthesis of CSPDA-NHS: 4-Cyano-4-[[(dodecylthio)thionylmethyl]thio]valeric acid (CSPDA RAFT, 500 mg, 1.25 mmol, 1 eq), N-hydroxysuccinimide (NHS, 150 mg, 1.5 mmol, 1.2 eq), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, 286.5 mg, 1.5 mmol, 1.2 eq) were added to a 25 mL round-bottom flask, followed by 10 mL of ultra-dry dichloromethane. Nitrogen gas was then introduced into the reaction flask for 15 minutes, and the mixture was stirred overnight at room temperature. The resulting reaction solution was rotary evaporated and then separated by silica gel column chromatography using ethyl acetate / petroleum ether (1:4, v / v) to yield 400 mg of the yellow product, CSPDA-NHS, in 63.7% yield.

[0083] (2) mPEG 2000 -CSPDA synthesis: mPEG 2000 -NH2 (500 mg, 0.25 mmol, 1 eq) and CSPDA-NHS (150.6 mg, 0.30 mmol, 1.2 eq) were added to a 25 mL round-bottom flask. 10 mL of ultra-dry dichloroethane was added, and the reaction mixture was purged with nitrogen. Triethylamine (TEA, 30 mg, 41 μL, 0.30 mmol, 1.2 eq) was added under nitrogen atmosphere, and the reaction was stirred overnight at room temperature. The resulting reaction mixture was rotary evaporated and then separated by silica gel column chromatography using dichloroethane / methanol (10:1, v / v) to obtain 250 mg of a yellow solid product, i.e., mPEG. 2000 -CSPDA, yield 41.7%.

[0084] II. Polyethylene Glycol - b - Polystyrene (mPEG) 2000 - b Synthesis of -PS):

[0085] Taking a degree of polymerization of 100 as an example, mPEG 2000 -CSPDA (50 mg, 0.021 mmol), styrene (218 mg, 2.1 mmol), DP n = 100), azobisisobutyronitrile (3.4 mg, 0.021 mmol) was added to a 10 mL round-bottom flask, along with 2.446 g of ultra-dry 1,4-dioxane. Nitrogen gas was injected into the reaction flask for 15 minutes, and the mixture was heated and stirred at 70 °C for 24 hours. After the reaction, the mixture was cooled and precipitated with excess methanol to obtain the purified polymer (M). n sec = 5500g·mol -1M W sec =6900 g·mol -1 , Ð = 1.26).

[0086] mPEG 2000 - b -Structural characterization map of PS as follows Figure 1 and Figure 2 As shown.

[0087] Example 2 This embodiment provides an amphiphilic polymer mPEG. n - b -PHPMA m The preparation method is described in Example 1, except that the styrene (water-repellent monomer) in step two is replaced with hydroxypropyl methacrylate (HPMA).

[0088] Example 3 This embodiment provides an amphiphilic polymer mPEG. n - b -PNAP m The preparation method is described in Example 1, except that the styrene (water-repellent monomer) in step two is replaced with ethylene naphthalene (NAP).

[0089] Example 4 This embodiment provides an amphiphilic polymer mPEG. n - b -PBP m The preparation method is described in Example 1, except that the styrene (water-repellent monomer) in step two is replaced with vinyl biphenyl (BP).

[0090] Example 5 This embodiment provides an amphiphilic polymer mPEG. n - b -PTPE m The preparation method is described in Example 1, except that the styrene (water-repellent monomer) in step two is replaced with (2-(4-vinylphenyl)ethylene-1,1,2-triyl)triphenyl (VTPE).

[0091] Example 6 This embodiment provides an amphiphilic polymer mPEG. n - b -PSMA m The preparation method is described in Example 1, except that the styrene (water-repellent monomer) in step two is replaced with octadecyl methacrylate (SMA).

[0092] Example 7 This embodiment provides an amphiphilic polymer mPEG. n - b -PSMA m The preparation method is described in Example 1, except that the styrene (water-repellent monomer) in step two is replaced with 4-bromostyrene (SBr).

[0093] Example 8 This embodiment provides an amphiphilic polymer mPEG. n - b -PYE m The preparation method is described in Example 1, except that the styrene (water-repellent monomer) in step two is replaced with vinylpyrene (YE).

[0094] Example 9 This embodiment provides an amphiphilic polymer mPEG. n - b -PBzMA m The preparation method is described in Example 1, except that the styrene (water-repellent monomer) in step two is replaced with benzyl methacrylate (BzMA).

[0095] Example 10 This embodiment provides a deuterated amphiphilic polymer mDPEG. n - b -PDS m The preparation method is the same as in Example 1, the only difference being the addition of mPEG in step one. 2000 -NH2 replaced with mDPEG 2000 -NH2 and replace the styrene (water-repellent monomer) in step two with deuterated styrene (DS).

[0096] Example 11 This embodiment provides an amphiphilic polymer, POEGMA. n - b -PS m The specific preparation method is as follows: I. Synthesis of poly(ethylene glycol) oligomethyl methacrylate (POEGMA) as the hydrophilic segment: Oligoethylene glycol methacrylate (OEGMA) 500 (4 g, 10 mmol, 72 eq), CSPDA RAFT (56 mg, 0.139 mmol, 1 eq), and azobisisobutyronitrile (2.3 mg, 0.139 mmol, 1 eq) were added to a round-bottom flask containing 5 mL of toluene. Nitrogen gas was introduced into the reaction flask for 15 minutes, and the mixture was heated and stirred at 70 °C for 16 hours. After cooling, the mixture was precipitated with diethyl ether, and the purified polymer, POEGMA-CSPDA (M), was obtained by rotary evaporation.n sec = 21700 g·mol -1 M W sec = 30100g·mol -1 , Ð = 1.39).

[0097] II. Poly(methacrylic acid) oligomeric glycol-β-polystyrene (POEGMA) n - b -PS m Synthesis of ) Taking a degree of polymerization of 750 as an example, POEGMA-CSPDA (96 mg, 6.0 μmol) and styrene (450 mg, 4.33 mmol) were mixed. DP n = 721), azobisisobutyronitrile (1 mg, 6.0 μmol) was added to a 25 mL round-bottom flask, along with 4.9 g of ultradry methanol. Nitrogen gas was injected into the reaction flask for 15 minutes, and the mixture was heated and stirred at 65 °C for 24 hours. After the reaction, the mixture was cooled, dialyzed against methanol, and then evaporated to dryness to obtain the purified polymer, POEGMA. n - b -PS m (M) n sec = 37200 g·mol -1 M W sec = 73300 g·mol -1 , Ð = 1.97).

[0098] POEGMA n - b -PS m Structural characterization maps such as Figure 3 and Figure 4 As shown.

[0099] Example 12 This embodiment provides an amphiphilic polymer, POEGMA. n - b -PFS m The preparation method is described in Example 11, except that the styrene (water-repellent monomer) in step two is replaced with pentafluorostyrene (FS).

[0100] Example 13 This embodiment provides a deuterated amphiphilic polymer, POEGMA. n - b -PDS m The preparation method is described in Example 11, except that the styrene (water-repellent monomer) in step two is replaced with deuterated styrene (DS).

[0101] Example 14 This embodiment provides an amphiphilic polymer PDMA. n - b -PS m The specific preparation method is as follows: I. Synthesis of poly(N,N-dimethylacrylamide) (PDMA) as the hydrophilic segment: N,N-dimethylacrylamide (1.227 g, 12 mmol, 50 eq), CSPDA RAFT (100 mg, 0.248 mmol, 1 eq), and azobisisobutyronitrile (24 mg, 0.1488 mmol, 0.6 eq) were added to a round-bottom flask containing 12.4 mL of ultra-dry dimethyl sulfoxide. Nitrogen gas was introduced into the reaction flask for 15 minutes, and the mixture was heated and stirred at 70 °C for 16 hours. After the reaction, the mixture was cooled, dialyzed against methanol, evaporated to dryness, and then evaporated to dryness again to obtain the purified polymer, PDMA (M... n sec = 4700 g·mol -1 M W sec = 5100 g·mol -1 , Ð = 1.07).

[0102] II. Poly(N,N-dimethylacrylamide)-b-polyethylene (PDMA) n - b -PS m Synthesis of )

[0103] Taking a degree of polymerization of 100 as an example, PDMA (200 mg, 0.029 mmol) and styrene (301.6 mg, 2.9 mmol) were mixed. DP n =100), azobisisobutyronitrile (4.7 mg, 0.029 mmol) was added to a 10 mL round-bottom flask, along with 4.6 g of ultra-dry 1,4-dioxane. Nitrogen gas was injected into the reaction flask for 15 minutes, and the mixture was heated and stirred at 70 °C for 24 hours. After cooling, the mixture was precipitated with methanol to obtain the purified polymer (M). n sec = 6500 g·mol -1 M w sec = 8200 g·mol -1 , Ð = 1.27).

[0104] PDMA n - b -PS m Structural characterization maps such as Figure 5 and Figure 6 As shown.

[0105] Example 15 This embodiment provides an NIR-II fluorescent molecule, the preparation method of which is as follows:

[0106] (1) Synthesis of compound 2: 1-(4-bromophenyl)-1,2,2-triphenylene (2.05 g, 5 mmol), substituted aniline (6.5 mmol), and tri-tert-butylphosphine (P( t -Bu)3 (16.2 mg, 0.08 mmol), Pd2(dba)3 (64 mg, 0.07 mmol) and sodium tert-butoxide (KO) t -Bu (625 mg, 6.5 mmol) was added to dry toluene (30 mL) and refluxed at 110 °C for 24 h under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, the solvent was evaporated under reduced pressure, and then water (30 mL) and chloroform (200 mL) were added. The organic phase was separated, washed with saturated brine, dried over anhydrous MgSO4, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography with n-hexane / chloroform (v / v = 5 / 1) as eluent to give compound 2. ¹H NMR (500 MHz, Methylene Chloride-d2) δ 7.23 (dd, J = 8.5, 7.2 Hz, 2H), 7.18–6.98 (m, 17H), 6.93–6.87 (m, 3H), 6.83–6.78 (m, 2H), 5.77 (s, 1H).

[0107] (2) Synthesis of compound 3: Compound 2 (8.9 mmol), 4-bromoiodobenzene (2.97 g, 10.6 mmol), and 1,10-phenanthroline (0.27 g, 1.5 mmol) were dissolved in toluene (30 mL). The mixture was heated to 100 °C, and then cuprous iodide (CuI, 0.15 g, 1.5 mmol) and KOH (1.23 g, 22.0 mmol) were added under nitrogen purging. The reaction mixture was refluxed at 120 °C for 48 h. After the reaction was completed, the mixture was cooled to room temperature, washed three times with water (50 mL), and the organic phase was dried over anhydrous Na₂SO₄. After removing the solvent, the residue was purified by silica gel column chromatography using ethyl acetate / petroleum ether (v / v = 1 / 40) as the eluent to give compound 3. ¹H NMR (500 MHz, Methylene Chloride-d2) δ 7.84 (d, J = 8.8 Hz, 2H), 7.82–7.74 (m, 2H), 7.74–7.52 (m, 18H), 7.43 (dd, J = 8.7, 3.7 Hz, 4H), 7.32 (d, J = 8.6 Hz, 2H).

[0108] (3) Synthesis of compound 4: Compound 3 (2.00 g, 3.46 mmol), bis(pinacol)diboron (1.93 g, 7.61 mmol), Pd(dppf)₂Cl₂ (119.3 mg, 0.16 mmol), and KOAc (1.02 g, 10.2 mmol) were added to a two-necked flask, and 1,4-dioxane was added as a solvent under nitrogen protection. The reaction system was reacted at 90 °C for 12 h. The post-reaction treatment was the same as that for compound 1. The crude product was purified by silica gel column chromatography using petroleum ether / dichloromethane (v / v = 3 / 1) as the eluent to obtain a yellow powdery product, namely compound 4 (1.25 g, yield 61.4%). ¹H NMR (400 MHz, Chloroform-d) δ 7.57 (dd, J = 8.5, 1.9 Hz, 2H), 7.15 (d, J = 7.7 Hz, 2H), 7.07–6.86 (m, 20H), 6.84–6.77 (m, 2H), 6.73 (d, J = 7.9 Hz, 2H), 1.26 (d, J = 1.6 Hz, 12H).

[0109] (4) Synthesis of TTT6,2-B: The synthesis of TTT6,2-B was performed according to existing literature (DOI: 10.1021 / jacs.2c07443 and 10.1002 / anie.201916357). Briefly, compound 4 (1 mmol), compound 5 (0.4 mmol), Pd(P(Ph)3)4 (0.025 mmol), toluene (1.5 mL), and water (0.3 mL) were added sequentially to a reaction tube and sealed under nitrogen protection. The reaction system was then heated to 130 °C in an oil bath and stirred for 8 h. After the reaction, the crude product was quenched and extracted with DCM. The obtained product was purified by column chromatography, finally yielding a dark green solid, namely TTT6,2-B, in 40% yield. The purified powder was dissolved in DMSO, and the solvent was slowly evaporated at room temperature to obtain single crystals for subsequent single-crystal structure analysis. The obtained product was structurally characterized by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HMR), with the following data: ¹H NMR (400 MHz, Methylene Chloride-d2) δ 7.48 (d, J = 8.7 Hz, 4H), 7.26–7.15 (m, 6H), 7.13–6.92 (m, 42H), 6.84 (d, J = 8.6 Hz, 4H), 6.76 (d, J = 8.6 Hz, 4H), 2.47 (d, J = 7.1 Hz, 4H), 1.26 (s, 2H), 1.05–0.65 (m, 16H), 0.56 (t, J = 6.8 Hz, 6H), 0.40 (t, J = 7.3 Hz, 6H). ¹³C NMR (400 MHz, Methylene Chloride-d2) δ 153.24, 147.43, 147.21, 146.07, 145.64, 144.79, 143.99, 143.82, 143.60, 140.79, 140.69, 138.67, 132.17, 131.27, 131.25, 131.22, 129.29, 128.93, 128 .08, 127.63, 127.60, 126.51, 126.42, 126.38, 126.31, 125.34, 124.68, 123. 40, 123.30, 116.23, 40.50, 34.37, 32.46, 28.55, 25.63, 22.81, 13.79, 10.41. HRMS(ESI):[M+H] + Theoretical value (C) 106 H 92 The value of N6S4 is 1576.62663, while the measured value is 1576.62572.

[0110] The structural characterization map of TTT6,2-B is as follows: Figures 7 to 9 As shown.

[0111] Example 16 This embodiment provides a nanoparticle PS-NPs (or TTT6,2-B-PS) with fluorescence enhancement properties, and its preparation method is as follows: 0.5 mg of the NIR-II fluorescent molecule (TTT6,2-B) prepared in Example 15 and 5 mg of the amphiphilic polymer mPEG prepared in Example 1 were used. n - b -PS m Dissolved in 1 mL of THF, the mixture was slowly added dropwise to 9 mL of ultrapure water under ultrasonic conditions in a water bath. After the addition was complete, ultrasonication was continued for two minutes. The prepared nanoparticle solution was transferred to a dialysis bag (molecular weight cutoff MWCO = 1000 Da). After dialysis, the resulting solution was filtered through a 0.45 μm needle filter. Finally, the nanoparticle solution was concentrated by ultrafiltration to obtain nanoparticles (PS-NPs).

[0112] mPEG n - b -PS m The preparation method for nanoparticles loaded with other NIR-II fluorescent molecules (4TPE-4N, TBBSD, 4TPA-TQ, TTBI, TPABT-TQ) is the same, except that TTT6,2-B is replaced with the corresponding other NIR-II fluorescent molecules.

[0113] Example 17 This embodiment provides a nanoparticle with fluorescence enhancement properties. The preparation method is the same as in Example 16, except that the amphiphilic polymer mPEG prepared in Example 1 is used instead. n - b -PS m Replace with the amphiphilic polymer mPEG prepared in Example 5. n - b -PTPE m .

[0114] Example 18 This embodiment provides a nanoparticle with fluorescence enhancement properties. The preparation method is the same as in Example 16, except that the amphiphilic polymer mPEG prepared in Example 1 is used instead. n - b -PS m Replace with the amphiphilic polymer POEGMA prepared in Example 11. n - b -PS m .

[0115] Example 19 This embodiment provides a nanoparticle with fluorescence enhancement properties. The preparation method is the same as in Example 16, except that the amphiphilic polymer mPEG prepared in Example 1 is used instead. n - b -PS m Replace with the amphiphilic polymer PDMA prepared in Example 14. n - b -PS m .

[0116] Comparative Example 1 This comparative example provides nanoparticles DSPE-NPs (or denoted as TTT6,2-B-DSPE), the preparation method of which is the same as in Example 16, the only difference being the use of the amphiphilic polymer mPEG prepared in Example 1. n - b -PS m Replace with the common amphiphilic carrier distearylphosphatidylethanolamine-block-polyethylene glycol (mPEG- b -DSPE).

[0117] Among them, mPEG- b The specific structure of DSPE is as follows: .

[0118] mPEG- b The preparation method of nanoparticles containing other NIR-II fluorescent molecules (4TPE-4N, TBBSD, 4TPA-TQ, TTBI, TPABT-TQ) via DSPE is the same, except that TTT6,2-B is replaced with the corresponding other NIR-II fluorescent molecules.

[0119] Comparative Example 2 This comparative example provides a nanoparticle, the preparation method of which is the same as in Example 16, the only difference being that the amphiphilic polymer mPEG prepared in Example 1 is used. n - b -PS m Replace it with a common amphiphilic carrier, polyethylene oxide-propylene oxide-ethylene oxide triblock copolymer (F127).

[0120] The specific structure of the F127 is as follows: .

[0121] The preparation method of F127 nanoparticles encapsulating other NIR-II fluorescent molecules (4TPE-4N, TBBSD, 4TPA-TQ, TTBI, TPABT-TQ) is the same, except that TTT6,2-B is replaced with the corresponding other NIR-II fluorescent molecules.

[0122] Comparative Example 3 This comparative example provides a nanoparticle, the preparation method of which is the same as in Example 16, the only difference being that the amphiphilic polymer mPEG prepared in Example 1 is used. n - b -PS m Replace with the common amphiphilic carrier polycaprolactone-block-polyethylene glycol (mPEG-) b -PCL).

[0123] Among them, mPEG- b The specific structure of PCL is as follows: .

[0124] mPEG- b The preparation method for PCL-encapsulated nanoparticles containing other NIR-II fluorescent molecules (4TPE-4N, TBBSD, 4TPA-TQ, TTBI, TPABT-TQ) is the same, except that TTT6,2-B is replaced with the corresponding other NIR-II fluorescent molecules.

[0125] Comparative Example 4 This comparative example provides a nanoparticle, the preparation method of which is the same as in Example 16, the only difference being that the amphiphilic polymer mPEG prepared in Example 1 is used. n - b -PS m Replace with the common amphiphilic carrier polylactic acid-glycolic acid copolymer-polyethylene glycol (mPEG-) b -PLGA).

[0126] Among them, mPEG- b The specific structure of PLGA is as follows: .

[0127] mPEG- b The preparation method of PLGA-encapsulated nanoparticles containing other NIR-II fluorescent molecules (4TPE-4N, TBBSD, 4TPA-TQ, TTBI, TPABT-TQ) is the same, except that TTT6,2-B is replaced with the corresponding other NIR-II fluorescent molecules.

[0128] The performance of the nanoparticles prepared in the above embodiments is then tested.

[0129] I. Storage and photostability tests of nanoparticles The particle size distribution and absorption stability of PS-NPs in water and PBS containing 10% FBS were periodically monitored using DLS and UV absorption spectroscopy, respectively, with a test cycle of 15 days. Aqueous solutions of PS-NPs and indocyanine green (ICG) (both 10 μM) were irradiated with an 808 nm laser (1 W / cm²) for 1 min each time, repeated for 15 cycles. After each irradiation, their UV-vis absorption and fluorescence spectra were collected. The maximum absorption peak (A0) and fluorescence emission peak (I0) of the unirradiated sample were used as the benchmark for normalized calculations.

[0130] II. Measurement of Fluorescent Quantum Yield (QY) The relative quantum yield (QY) of the dye was determined using IR-26 (QY = 0.05%) as a reference. A series of solutions with absorbance intensities of approximately 0.02, 0.04, 0.06, 0.08, and 0.10 at 808 nm were prepared by diluting IR-26 in 1,2-dichloroethane. Photoluminescence (PL) spectra were recorded under 808 nm light excitation, and the emission spectra were integrated in the range of 850–1500 nm. The same procedure was applied to the dye samples in water. Subsequently, the integrated emission intensity was plotted against the corresponding absorbance values, and a linear fit was performed. QY was calculated according to formula (1): QY 样本 = QY 参考 · S 样本 / S 参考 ·(n 样本 / n 参考 )^2; Among them, QY 样本 It is the relative quantum yield of nanoparticles, QY 参考 The quantum yield of IR-26 (approximately 0.05% in dichloroethane), S 样本 and S 参考 The slopes are obtained by linear fitting after plotting the integrated emission spectra of nanoparticles and IR-26 against the absorbance at 808 nm. 样本 and n 参考 These represent the refractive indices of water (1.333) and dichloroethane (1.413), respectively.

[0131] III. In vitro concentration-dependent fluorescence imaging Concentration-dependent fluorescence was evaluated in vitro using DSPE-NPs (H2O), PS-NPs (H2O), and PS-NPs (D2O) at concentrations ranging from 0.625 to 80 μM. Aliquots (300 μL) were placed in 96-well plates and imaged using an InGaAs camera (1000 nm long-pass filter, 3000 mA, 15 ms exposure). Fluorescence intensity was quantified using ImageJ software.

[0132] IV. Assessment of in vitro tissue penetration depth Fresh chicken breast slices were layered to construct a tissue simulation model with increasing thickness. PS-NPs solution (300 μL, 1 mM) was added to 96-well plates, with DSPE-NPs used as a control. NIR-II fluorescence decay with depth was monitored under 808 nm excitation (1000 nm long-pass filter, 8000 mA, 200 ms exposure), and intensity was quantified using ImageJ.

[0133] V. Signal-to-back ratio (SBR) and full width at half maximum (FWHM) To determine the signal-to-weight ratio (SBR) and fluorescence-to-weight ratio (FWHM) in NIR-II bioimaging, a standardized procedure was followed. First, a region of interest (ROI) was selected for each measurement. Within this ROI, a white dashed line was drawn, and the fluorescence intensity distribution curve crossing this line was acquired and fitted with a Gaussian function. Signal intensity I 信号 I is defined as the peak value of the fitted Gaussian curve, while the baseline intensity of the fitted curve is used as the background intensity. 背景 Calculate SBR=I 信号 / I 背景 For FWHM measurement, draw another white line within the ROI. Similarly, extract the intensity distribution along this line and perform Gaussian fitting. Then, determine the FWHM by the width of the fitted Gaussian curve at half its maximum height.

[0134] VI. Angiography at the NIR-II Window Mice (n = 3) were intravenously (iv) injected with either DSPE-NPs or PS-NPs (200 μL, 1 mM) for NIR-II vascular imaging. After anesthesia, the animals were imaged using a MASR in vivo imaging system at 808 nm excitation. Emissions were collected using long-pass (LP) filters at optimized exposure settings: 1000 nm LP, 8 ms; 1100 nm LP, 20 ms; 1200 nm LP, 80 ms; 1300 nm LP, 300 ms; 1400 nm LP, 2000 ms (laser current 8000 mA). Hind limb vessels (white-lined areas) were imaged across the entire LP filter series, while high-resolution images of abdominal and cerebral microvessels were acquired at 1400 nm LP. Quantitative analyses (fluorescence intensity, Gaussian fit, SBR, and FWHM) were performed using ImageJ and Origin 2019b software.

[0135] VII. NIR-II fluorescence imaging of tumors and image-guided surgery Female BALB / c mice (n = 3) were subcutaneously inoculated with 4T1 tumors (approximately 200 mm in size). 3 ; 1.5×10 6 Cells were suspended in 100 μL PBS and injected intravenously with either DSPE-NPs or PS-NPs (200 μL, 500 μM). NIR-II imaging (MARS system, 808 nm excitation, 3000 mA, 15 ms exposure, 1000 nm LP) was performed at 0–168 hours (0, 0.5, 1, 3, 6, 9, 12, 24, 36, 48, 72, 96, 120, 168 hours). On day 7, tumors and major organs (heart, liver, spleen, lung, kidney) were removed for ex vivo imaging. ImageJ quantitative fluorescence was used.

[0136] In another independent experiment, PS-NPs (50 μL, 500 μM, dissolved in H2O or D2O) were injected intratumorally into 4T1 xenograft tumors and imaged (3000 mA, 20 ms exposure).

[0137] 8. NIR-II Guided Surgery By using CT26-Luc cells (50 μL, 2 × 10⁻⁶) 7An orthotopic colorectal cancer model was established by injecting PS-NPs (200 μL, 500 μM) into the colonic wall. Tumor growth was verified by bioluminescence. Mice were intravenously injected with PS-NPs. 48 hours later, the colorectal region was exposed and imaged (MARS system, 808 nm excitation, 3000 mA, 1000 nm LP). Grossly visible nodules were excised, followed by resection of residual lesions under real-time NIR-II imaging guidance. The excised nodules were measured and H&E stained.

[0138] Performance test results are as follows Figures 10 to 26 As shown.

[0139] In summary, the core innovation of this invention lies in "one type of polymer structure + two types of mechanisms of action + platformization effect", as detailed below: (1) A class of polymer structures: amphiphilic polymers with strong hydrophobic / rigid hydrophobic segments The core carrier of this invention is an amphiphilic polymer comprising a hydrophilic segment and a hydrophobic rigid segment (preferably strongly hydrophobic and possessing high chain rigidity / high Tg / aromatic structural characteristics). Through self-assembly, it forms assemblies such as micelles / nanoparticles to encapsulate NIR-II fluorescent molecules and construct a "dense hydrophobic microenvironment." The function of this type of carrier is not merely to achieve water dispersion, but rather to leverage the hydrophobic rigid segment to form a denser aggregate structure and a more effective interfacial barrier, thereby achieving significant brightening in the aqueous phase.

[0140] (2) One of the two mechanisms of action: rigid confinement inhibits the non-radiative decay of aggregated states. By strengthening the hydrophobic interactions and achieving a more compact co-assembly between the hydrophobic rigid segment and the NIR-II fluorescent molecule, the vibrational / rotational degrees of freedom within the molecule are confined, reducing nonradiative relaxation induced by intramolecular motion. Molecular dynamics evidence shows that the thermal motion (MSD) of NIR-II fluorescent molecules is more restricted in the PS microenvironment, and a wider range of energy barriers are imposed on the rotation of multiple benzene rings, demonstrating the role of "rigid confinement" in reducing molecular degrees of freedom.

[0141] (3) The second of the two mechanisms of action: interfacial isolation inhibits water-induced quenching The strongly hydrophobic segment drives the formation of a denser hydrophobic shell / interface layer, reducing quenching caused by interfacial water ingress and high vibrational coupling with OH groups, thereby improving aqueous / bulk luminescence efficiency. Evidence for this mechanism includes: The radial distribution function (RDF) indicates that the average distance between the key site of the NIR-II fluorescent molecule and the oxygen atom of the water molecule is significantly increased in the PS system (e.g., from 2.95 Å to 4.04 Å), showing increased spatial separation and weakened coupling between the molecule and water. Evidence for D2O brightening: Compared to H2O, QY in the PS system can be further improved (e.g., by 22.3%), reflecting the reduction of non-radiative channels after the weakening of interfacial vibrational coupling.

[0142] (4) Platform effect: It can universally enhance the brightness of multiple types of NIR-II fluorescent molecules, while it is "difficult to improve" the brightness of non-NIR-II carriers. The innovation of this invention does not lie in "optimizing a certain dye", but in providing a reusable "carrier microenvironment engineering" platform: for a variety of NIR-II fluorescent molecules with significant structural differences and large intrinsic QY ranges, strongly hydrophobic / rigid hydrophobic segment functionalized carriers can stably bring higher emission intensity and more significant QY improvement; in contrast, conventional non-such carriers have limited or no overall improvement.

[0143] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A nanoparticle having fluorescence-enhancing properties, characterized in that, include: NIR-II fluorescent molecules and amphiphilic polymers encapsulating the NIR-II fluorescent molecules; The amphiphilic polymer has a hydrophilic segment and a rigid hydrophobic segment.

2. The nanoparticles according to claim 1, characterized in that, The hydrophilic segment is selected from at least one of polyethylene glycol, methoxy polyethylene glycol, poly(N,N-dimethylacrylamide), polymethyl methacrylate oligoethylene glycol, polyglycerol monoacrylate and polyacrylamide or their deuterated derivatives.

3. The nanoparticles according to claim 1, characterized in that, The rigid hydrophobic segment is selected from at least one deuterated form of polystyrene, polyhydroxypropyl methacrylate, polyoctadecyl methacrylate, polyvinyl biphenyl, poly[1-(4-vinylphenyl)-1,2,2-triphenyl]ethylene, polybenzyl methacrylate, polyvinylnaphthalene, polyvinylpyrene, poly4-bromostyrene, and polypentafluorostyrene.

4. The nanoparticles according to claim 1, characterized in that, The amphiphilic polymer is selected from at least one of the following structures: ； In the structural formula, n and m represent the degree of aggregation, where n is an integer between 26 and 222, and m is an integer between 15 and 1260. b This is the block segment symbol in the amphiphilic polymer.

5. The nanoparticles according to claim 1, characterized in that, The amphiphilic polymer is selected from at least one of the following structures: ； In the structural formula, n represents the degree of aggregation, which is an integer between 51 and 144. b This is the block segment symbol in the amphiphilic polymer.

6. The nanoparticles according to claim 1, characterized in that, The NIR-II fluorescent molecule is selected from at least one of the following structures: ； R1 and R6 are independently selected from substituted or unsubstituted aryl, substituted or unsubstituted triphenylamino, substituted or unsubstituted tristyryl, and substituted or unsubstituted tetrastyryl. R2 and R5 are independently selected from hydrogen, halogen, C1-C30 straight-chain or branched alkyl, C1-C30 straight-chain or branched alkoxy, C3-C6 cycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted triphenylamine, substituted or unsubstituted tristyryl, substituted or unsubstituted tetrastyryl. R3 and R4 are independently selected from halogens, C1-C30 straight-chain or branched alkyl groups, C1-C30 straight-chain or branched alkoxy groups, C3-C6 cycloalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted triphenylamine groups, substituted or unsubstituted tristyryl groups, and substituted or unsubstituted tetrastyryl groups.

7. The nanoparticles according to claim 1, characterized in that, The NIR-II fluorescent molecule is selected from at least one of the following structures: 。 8. A method for preparing nanoparticles according to any one of claims 1-7, characterized in that, Including the following steps: The NIR-II fluorescent molecule and the amphiphilic polymer were dissolved in an organic solvent to obtain a mixed solution; The mixed solution was added dropwise to water under ultrasonic conditions to obtain nanoparticles.

9. The method for preparing nanoparticles according to claim 8, characterized in that, The organic solvent is selected from at least one of tetrahydrofuran, dimethyl sulfoxide, and dichloromethane.

10. The application of the nanoparticles as described in any one of claims 1-7 in fluorescence imaging.

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