Two-photon type AIE nano-particles and preparation method and application thereof

Abstract

A preparation method of the two-photon type AIE nano-particles comprises the following steps: S1, mixing tetrahydrofuran, a tetrahydrofuran solution containing an AIE material, a tetrahydrofuran solution containing poloxamer 407 and tetramethyl orthosilicate, and performing uniform treatment to obtain a mixed solution; s2, dropwise adding the mixed solution obtained in the step S1 into deionized water in stirring, then performing uniform treatment again, and then performing stirring; s3, dialyzing, and then filtering, so as to obtain purified two-photon type AIE nanoparticles; and S4, modifying glucosan after amino modification on the surface of the silicon dioxide to obtain the glucan-modified two-photon AIE nanoparticles. The preparation method disclosed by the invention has the advantages of simple preparation and low cost, and the obtained nanoparticles have high stability, high safety and high two-photon performance, and can be used for two-photon imaging of deep biological tissues.

Description

Technical Field

[0001] The present invention relates to the field of biochemistry, and in particular to a two-photon AIE nanoparticle and a preparation method and application thereof. Background Art

[0002] Aggregation-induced emission (AIE) is a recently emerging fluorescence generation technology. Fluorescent materials exhibiting AIE properties are non-luminescent or only weakly fluorescent when dispersed in dilute solutions, but exhibit significantly enhanced fluorescence at high concentrations or when aggregated in solids. Current research on the mechanism of AIE suggests that AIE molecules generally possess a unique propeller structure. When excited in a dispersed state, this propeller structure can rotate freely, dissipating radiative energy. In an aggregated state, molecular motion is restricted, significantly preventing non-radiative decay upon excitation, resulting in enhanced fluorescence.

[0003] Compared with traditional fluorescent materials, AIE materials have the following advantages: (1) high fluorescence quantum yield, which can be regulated by dispersion-aggregation; (2) excellent photostability and large Stokes shift; and (3) good biocompatibility. Currently, AIE nanoparticles (AIE NPs) have been widely used in biomedical applications such as vascular imaging and protein fluorescent labeling, and have important application value.

[0004] The classic preparation strategy for AIE NPs leverages the universal lipid solubility of AIE materials by encapsulating them with amphiphilic materials (such as poloxamer 407, also known as Pluronic F127) for self-assembly, resulting in nanomicelles encapsulated with the AIE material. The steps are summarized as follows: Pluronic F127 and the AIE material are mixed in a volatile organic solvent (such as tetrahydrofuran) and then added dropwise to water, allowing the Pluronic F127 to self-assemble into nanomicelles. During the nanomicelle formation process, the lipid-soluble AIE material is encapsulated within the hydrophobic core of the Pluronic F127 nanomicelles, while the outer rim of the micelles is formed by long hydrophilic chains, resulting in water-soluble AIE NPs. The solution is continuously stirred to evaporate the organic solvent, and further purification is achieved by filtration or centrifugation. ZheFeng et al. used the above strategy to encapsulate the AIE material 2FT-oCB in Pluronic F127 micelles. The resulting AIE NPs (2FT-oCB dots) can emit near-infrared (NIR) fluorescence when excited and were used for two-photon depth imaging of mouse uterine blood vessels (Document DOI: 10.1038 / s41467-023-40728-6); Xiaoming Yu et al. used a similar strategy to develop AIE NPs (2FT-oCB dots) and used two-photon microscopy to achieve uterine cavity imaging and uterine angiography, which has both biosafety and clinical practicality (Document DOI: 10.1016 / j.nantod.2021.101235).

[0005] As can be seen from the above, AIE materials can be encapsulated with amphiphilic materials to prepare AIE NPs. This strategy is convenient, simple, and effective, and has important application value in the field of bioluminescence imaging. However, the AIENPs prepared by this classic strategy have the structure of nanomicelles. When the micelle concentration falls below the critical micelle concentration (CMC) in the biological environment, they are prone to dissociation, resulting in leakage of the AIE material, the formation of fluorescent background interference, and even toxic side effects. In addition, when using two-photon technology for deep tissue imaging, AIE NPs must have good two-photon performance. The two-photon performance of micellar AIE NPs (such as fluorescence quantum efficiency and two-photon absorption cross section) needs to be further improved.

[0006] Therefore, studying how to improve the stability, fluorescence properties and biocompatibility of AIE NPs has important scientific significance and practical value. Summary of the Invention

[0007] To address the above problems, the present invention provides a two-photon AIE nanoparticle and its preparation method and application, which are used to prepare nanoparticles with high stability, high safety and high two-photon performance for two-photon imaging of deep biological tissues.

[0008] The present invention provides a method for preparing two-photon AIE nanoparticles, comprising the following steps: S1. Mixing tetrahydrofuran, a tetrahydrofuran solution containing an AIE material, a tetrahydrofuran solution containing poloxamer 407 (Pluronic F127), and methyl orthosilicate to obtain a mixed solution; S2 S1 obtained by adding the mixture dropwise to the stirring deionized water; S3. Dialysis and filtration were performed to obtain purified two-photon AIE nanoparticles AF@SiO2NPs.

[0009] Furthermore, after step S3, step S4 is also included: adding the two-photon AIE nanoparticles AF@SiO2NPs to hydrochloric acid and aminopropyltriethoxysilane while stirring, ultrafiltering twice after stirring, adding PBS to resuspend, adding to PBS containing N-hydroxysuccinimide activated ester-modified dextran, stirring in an ice bath, ultrafiltering, adding ultrapure water to resuspend, and ultrafiltering again to obtain purified two-photon AIE nanoparticles AF@SiO2-Dex NPs.

[0010] Furthermore, in step S4, the volume ratio of the two-photon AIE nanoparticles AF@SiO2NPs, the hydrochloric acid, the aminopropyltriethoxysilane, and the PBS containing N-hydroxysuccinimide activated ester-modified dextran is (180-220): (180-220): 3: (180-220), the concentration of the hydrochloric acid is 0.9-1.1 M, and the concentration of the dextran is 4.5-5.5 mM.

[0011] Furthermore, the specific operation of the ultrafiltration is: using an ultrafiltration centrifuge tube with a molecular weight cut-off of 100 kD and centrifuging at 5000 rpm for 18-22 minutes.

[0012] Further, hydrochloric acid and aminopropyltriethoxysilane were added, stirred for 22-26 h, and stirred in an ice-water bath for 10.8-13.2 h.

[0013] Furthermore, the volume ratio of tetrahydrofuran, the tetrahydrofuran solution containing the AIE material, the tetrahydrofuran solution containing poloxamer 407, and methyl orthosilicate in step S1 is (18-22):1:(18-22):(0.7-0.8), the concentration of the tetrahydrofuran solution containing the AIE material is 0.9-1.1 μM, and the concentration of the tetrahydrofuran solution containing poloxamer 407 is 45-55 mg / mL.

[0014] Furthermore, the AIE material is AF, named N,N'-((Benzo[c][1,2,5]thiadiazole-4,7-diylbis(thiophene-5,2-diyl))bis(4,1-phenyl))bis(N-phenylnaphthalen-1-amine), and the structure of AF is: .

[0015] Furthermore, the synthetic route of AF is as follows: Furthermore, the preparation method of AF comprises the following steps: (1) Dissolve (4-(naphthalen-1-yl(phenyl)amino)phenyl)boric acid in ethanol, add a toluene solution containing 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole and a sodium carbonate aqueous solution, and then perform deoxygenation treatment; (2) injecting a toluene solution containing tetrakis(triphenylphosphine)palladium(0) and performing deoxygenation treatment; (3) Stir vigorously at 85-95 °C for more than 24 hours under nitrogen protection, cool to room temperature, and rotary evaporate to obtain a crude product; (4) The crude product was dissolved in dichloromethane and recrystallized from n-hexane, and then purified by silica gel column chromatography to obtain the final product AF.

[0016] Furthermore, the equivalent ratio of (4-(naphthalen-1-yl(phenyl)amino)phenyl)boric acid, 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole and tetrakis(triphenylphosphine)palladium(0) is (18-22):(9-11):1.

[0017] Furthermore, the molar volume ratio of the (4-(naphthalen-1-yl(phenyl)amino)phenyl)boric acid to ethanol is 0.25 mmol / mL, the molar volume ratio of the 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole to toluene is 0.25 mmol / mL, the concentration of the sodium carbonate aqueous solution is 1.8-2.2 M, and the volume ratio of the toluene solution to the sodium carbonate aqueous solution is 5:(1.8-2.2).

[0018] Furthermore, the specific operation of the deoxygenation treatment is: vacuuming, filling with nitrogen after 10 minutes, and repeating this cycle 3 times.

[0019] Furthermore, the eluent used for the silica gel column chromatography purification is n-hexane and dichloromethane in a volume ratio of 2:1.

[0020] Furthermore, after the mixing in step S1 and the addition to the stirring deionized water in step S2, a uniform treatment is further included. Specifically, the uniform treatment is performed using an ultrasonic probe of a cell disruptor with a power of 50 W for 10 minutes and an interval of 10 seconds on / 10 seconds off.

[0021] Furthermore, the stirring speed of the deionized water in step S2 is 1500 rpm, and the stirring speed after the uniform treatment in step S2 is 1500 rpm, and the duration is 5 days.

[0022] Furthermore, the step S2 further includes the following steps after the uniform treatment and before the stirring: transferring the solution into a container, covering the bottle mouth with tin foil, and using a toothpick to pierce a plurality of small holes.

[0023] Furthermore, the specific operation of dialysis in step S3 is: transferring the solution obtained in S2 into a dialysis bag with a molecular weight cut-off of 40K, and dialyzing for 3 days.

[0024] Furthermore, the specific operation of filtering in step S3 is: filtering using a water-based syringe filter with a pore size of 0.45 μm, and then filtering using a water-based syringe filter with a pore size of 0.22 μm.

[0025] The present invention also provides a two-photon AIE nanoparticle, which is prepared by the above-mentioned preparation method.

[0026] The present invention also provides the use of the above-mentioned two-photon AIE nanoparticles in deep imaging of biological tissues.

[0027] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for preparing two-photon AIE nanoparticles AF@SiO2NPs and AF@SiO2-Dex NPs, which have the advantages of simple preparation and low cost. The prepared nanoparticles have the following beneficial effects: (1) Enhanced stability of nanoparticles: Compared with ordinary micellar AIE nanoparticles (AF@F127 NPs), the nanoparticles AF@SiO2NPs and AF@SiO2-Dex NPs are characterized by the formation of a silica layer on the outer edge of the hydrophobic core of the AIE micelles. The silica layer of the obtained nanoparticles AF@SiO2NPs and AF@SiO2-Dex NPs can effectively inhibit the dissociation of the micelles, thereby enhancing the stability of the nanoparticles and inhibiting the leakage of AIE materials. In a long-term (1 month) static experiment, they have better stability than the nanoparticles AF@F127 NPs prepared based on the classical strategy (simply using poloxamer 407 without generating a silica layer).

[0028] (2) Enhanced biocompatibility of nanoparticles: The silica layer of AF@SiO2NPs can be easily modified by hydrolysis of silicon-based materials to achieve engineering modification; further surface modification of AF@SiO2NPs with dextran makes the nanoparticles electrically neutral as a whole, which helps to reduce the adsorption of blood proteins, reduce the immunogenicity of nanoparticles, and further improve biocompatibility. The obtained AF@SiO2-Dex NPs showed no obvious toxicity in both cell experiments and animal experiments, and had no significant effect on cell morphology and survival rate; in a 2-week biocompatibility experiment of mice injected with nanoparticles AF@SiO2-Dex NPs through the tail vein, it was found that the nanoparticles did not cause significant developmental inhibition, and had no significant effect on blood and organ function.

[0029] (3) Enhanced two-photon fluorescence performance of nanoparticles: The present invention constructs a silica layer on the outer edge of the AIE hydrophobic core of the micellar nanoparticles. The hydrophobic core of the obtained nanoparticles AF@SiO2NPs and AF@SiO2-Dex NPs where the AIE material AF is located is squeezed by the centripetal force of the silica layer, which greatly enhances the aggregation-induced emission effect of the AIE material AF. Compared with the nanoparticles AF@F127NPs prepared based on the classical strategy (using only poloxamer 407 without generating a silica layer), the two-photon fluorescence properties of AF@SiO2NPs and AF@SiO2-DexNPs, such as fluorescence quantum efficiency and two-photon absorption cross-section, have been significantly improved: the fluorescence quantum efficiency of AF@F127 NPs is 12.23±1.95%, while the fluorescence quantum efficiency of AF@SiO2NPs and AF@SiO2-DexNPs are 48.76±3.09% and 45.90±2.39%, respectively; the two-photon absorption cross-section of AF@F127 NPs at the 2nd absorption peak (1060nm band) is 49.63±8.48 GM, while that of AF@SiO2NPs and AF@SiO2-Dex NPs are 266.56±14.72 GM and 256.08±10.97 GM, respectively. The enhancement of fluorescence efficiency and two-photon absorption cross-section makes the nanoparticles AF@SiO2NPs and AF@SiO2-Dex NPs have better excitation efficiency after two-photon excitation, which is beneficial to reducing the required excitation light power in actual two-photon imaging applications, significantly reducing the phototoxicity of excitation light to living tissues, extending the observation time and improving experimental safety.

[0030] (4) Nanoparticles are suitable for two-photon vascular imaging: In a two-photon imaging experiment on living mice, nanoparticles AF@SiO2-Dex NPs were injected into the mouse's blood circulation via the tail vein. The mouse uterine vascular network was visualized using a two-photon microscope, with an imaging depth of more than 500 μm. The three-dimensional reconstruction of the vascular network was performed using an optical sectioning sequence. AF@SiO2-Dex NPs were co-imaged with FITC-dextran, confirming that AF@SiO2-Dex NPs can reside in the blood vessels for a longer time. The modification of dextran can reduce the immunogenicity of the nanoparticles and effectively prolong their residence time in the blood circulation. While imaging blood vessels with nanoparticles AF@SiO2-Dex NPs, the diffusion of FITC-dextran in the tissue was observed, suggesting that they can be used to study vascular permeability. The above results indicate that two-photon AIE nanoparticles AF@SiO2-DexNPs have good two-photon deep tissue imaging performance and have good application potential in biofluorescence imaging and labeling. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0032] Figure 1 The structural diagram of nanoparticles AF@SiO2NPs; Figure 2 The synthetic route of nanoparticles AF@SiO2NPs; Figure 3 The structural diagram of nanoparticles AF@SiO2-Dex NPs; Figure 4 The synthetic route of nanoparticles AF@SiO2-Dex NPs; Figure 5 is the fluorescence characteristic diagram of AIE material AF; Figure 6 This is the aggregation-induced emission characteristic diagram of the AIE material AF; Figure 7 The optimized geometric molecular structure and dihedral angle distribution of AIE material AF; Figure 8 H NMR spectra of nanoparticles AF@F127 NPs, AF@SiO2 NPs, AF@SiO2-Dex NPs, and Dextran-NHS; Figure 9 UV-visible absorption spectra of nanoparticles AF@F127 NPs and AF@SiO2NPs; Figure 10 is the fluorescence emission spectra of nanoparticles AF@F127 NPs and AF@SiO2NPs; Figure 11 DLS particle size distribution diagram of nanoparticles AF@F127 NPs and AF@SiO2NPs; Figure 12 TEM morphology of nanoparticles AF@F127 NPs; Figure 13 TEM morphology of nanoparticles AF@SiO2NPs; Figure 14 Zeta potential distribution diagram of nanoparticles AF@F127 NPs and AF@SiO2NPs; Figure 15 The photos taken with white light and fluorescence of AF@SiO2NPs nanoparticles with different amounts of TMOS added; Figure 16The DLS particle size change diagram of nanoparticles AF@SiO2NPs with different amounts of TMOS added; Figure 17 TEM morphology of AF@SiO2NPs with different amounts (40µL and 60µL) of TMOS added (the scale bar is 50 nm); Figure 18 The fluorescence enhancement factor of AF@SiO2NPs nanoparticles with different amounts of TMOS added; Figure 19 The fluorescence quantum efficiency diagram of nanoparticles AF@F127 NPs and AF@SiO2NPs; Figure 20 Two-photon excitation images of nanoparticles AF@F127 NPs, AF@SiO2 NPs, and reference rhodamine B (RB) loaded into capillary glass tubes (scale bar: 100 μm); Figure 21 The logarithmic distribution diagram of two-photon excitation power and fluorescence intensity of nanoparticles AF@F127 NPs and AF@SiO2NPs; Figure 22 is the two-photon absorption cross section of nanoparticles AF@F127 NPs and AF@SiO2NPs at different wavelengths; Figure 23 TEM morphology of nanoparticles AF@SiO2-Dex NPs; Figure 24 The fluorescence characteristics of nanoparticles AF@SiO2-Dex NPs; Figure 25 is the DLS particle size distribution diagram of nanoparticles AF@SiO2-Dex NPs; Figure 26 Zeta potential distribution diagram of nanoparticles AF@SiO2NPs and AF@SiO2-Dex NPs; Figure 27 The fluorescence intensity of nanoparticles AF@F127 NPs, AF@SiO2NPs and AF@SiO2-Dex NPs changes with time; Figure 28 The results of the toxicity study of nanoparticles AF@SiO2-Dex NPs on HeLa cells and HUVEC cells; Figure 29 Figure 2 is the weight change of mice after adding different concentrations of nanoparticles AF@SiO2-Dex NPs; Figure 30 The organ coefficients of different organs with different concentrations of nanoparticles AF@SiO2-Dex NPs added; Figure 31Blood biochemical indicators after adding different concentrations of nanoparticles AF@SiO2-Dex NPs; Figure 32 Pathological analysis of major organs after adding different concentrations of nanoparticles AF@SiO2-Dex NPs (scale bar is 50 μm); Figure 33 In vivo uterine vascular imaging of mice using AF@SiO2-Dex NPs (scale bar: 100 μm). Figure 34 Fluorescence imaging of mice 10 minutes after co-injection of nanoparticles AF@SiO2-Dex NPs and commercial FITC dextran into the tail vein (scale bar: 100 μm); Figure 35 Linear scan fluorescence intensity spectrum of mice 10 minutes after co-injection of nanoparticles AF@SiO2-Dex NPs and commercial FITC dextran into the tail vein; Figure 36 Images of mice 60 minutes after co-injection of AF@SiO2-Dex NPs and commercial FITC-dextran into the tail vein (scale bar: 100 μm); Figure 37 Linear scan fluorescence intensity spectrum of mice 60 minutes after co-injection of nanoparticles AF@SiO2-Dex NPs and commercial FITC dextran into the tail vein. DETAILED DESCRIPTION

[0033] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the present invention. The present invention is described in detail below with reference to the specific embodiments.

[0034] The present invention provides a method for preparing two-photon AIE nanoparticles (AF@SiO2NPs), the structure of which is as follows: Figure 1 As shown in Figure 1, the AIE material AF is a newly developed AIE material with excellent AIE properties. Upon excitation, it emits red fluorescence in the near-infrared region 1. The nanoparticles AF@SiO2NPs made from the AIE material AF have an absorption peak (530 nm) and a fluorescence emission peak (710 nm) similar to those of the AF small molecule. The fluorescence emission peak of the nanoparticles AF@SiO2NPs is in the near-infrared region 1, which has good penetration depth in imaging applications. The synthesis route is shown in Figure 1. Figure 2 shown.

[0035] Specifically, the preparation method comprises the following steps: S1. Mix ultra-dry tetrahydrofuran (THF) (containing less than 30 ppm of water), a THF solution containing an AIE material, a THF solution containing poloxamer 407, and tetramethyl orthosilicate (TMOS), and homogenize to obtain a mixed solution. S2 S1 obtained by adding the mixture dropwise to the stirring deionized water, and then again do the uniform treatment, and then stir; S3. Dialysis followed by filtration yielded purified two-photon AIE nanoparticles (AF@SiO2NPs).

[0036] Principle of the Invention: Silica possesses extremely high mechanical strength and biochemical inertness. To enhance the stability of AIE nanoparticles, a silica encapsulation layer is constructed around the hydrophobic core of the micelle, which contains the AIE material. This encapsulation effectively encapsulates the hydrophobic core, thereby inhibiting degradation of the micelle in the biological environment and preventing leakage of the AIE material. Furthermore, the silica encapsulation of the AIE material further strengthens the centripetal force exerted on the hydrophobic core, enhancing the aggregation-induced emission effect and thus improving the two-photon fluorescence properties of the AIE nanoparticles, such as fluorescence quantum efficiency and two-photon absorption cross section, facilitating two-photon imaging of living tissues.

[0037] In other embodiments, other AIE materials can be selected to utilize silica encapsulation strategies to prepare fluorescent nanoparticles with enhanced stability and aggregation-induced emission (AIE) effects. Furthermore, silicon sources can be introduced into the outer surface of the hydrophobic core silica layer to further modify the surface and achieve various functionalizations.

[0038] Specifically, after step S3, step S4 is further included. Step S4 is to further modify the surface of the silica core with dextran, which can further improve the biocompatibility of the nanoparticles and reduce the immunogenicity, thereby achieving a long-term residence time in the blood circulation. After step S4, two-photon AIE nanoparticles AF@SiO2-Dex NPs are prepared. The structure of AF@SiO2-Dex NPs is as follows: Figure 3 The synthetic route of AF@SiO2-Dex NPs is shown in Figure 4 shown.

[0039] Specifically, the specific operation of step S4 is as follows: the two-photon AIE nanoparticles AF@SiO2NPs are added to hydrochloric acid and aminopropyltriethoxysilane (APTES) while stirring, and after stirring, they are ultrafiltered twice, resuspended in PBS, and added to PBS containing N-hydroxysuccinimide activated ester (NHS)-modified dextran, stirred in an ice bath, ultrafiltered, resuspended in ultrapure water, and ultrafiltered again to obtain purified two-photon AIE nanoparticles AF@SiO2-Dex NPs.

[0040] Specifically, it is characterized in that the volume ratio of the two-photon AIE nanoparticles AF@SiO2NPs, the hydrochloric acid, the aminopropyltriethoxysilane, and the PBS containing N-hydroxysuccinimide activated ester-modified dextran in step S4 is (180-220): (180-220): 3: (180-220), preferably 200:200:3:200, the concentration of the hydrochloric acid is 0.9-1.1 M, preferably 1 M, and the concentration of the dextran is 4.5-5.5 mM, preferably 5 mM.

[0041] Specifically, the molecular weight of the dextran is 1.35-1.65K, preferably 1.5K.

[0042] Specifically, the ultrafiltration operation is: using an ultrafiltration centrifuge tube with a molecular weight cut-off of 100 kD and centrifuging at 5000 rpm for 18-22 minutes, preferably 20 minutes.

[0043] Specifically, hydrochloric acid and aminopropyltriethoxysilane are added, stirred for 22-26 h, preferably 24 h, and stirred in an ice-water bath for 10.8-13.2 h, preferably 12 h.

[0044] Specifically, by surface modification of AF@SiO2-Dex NPs, such as using 3-aminopropyltriethoxysilane (APTES) to modify the outer surface of the silica layer, two-photon AIE nanoparticles with amino groups on the surface can be prepared for further modification with other chemical groups (targeting or fluorescence) or proteins.

[0045] Specifically, the volume ratio of the ultra-dry tetrahydrofuran in step S1, the tetrahydrofuran solution containing the AIE material, the tetrahydrofuran solution containing poloxamer 407, and methyl orthosilicate is (18-22):1:(18-22):(0.7-0.8), the concentration of the tetrahydrofuran solution containing the AIE material is 0.9-1.1 μM, and the concentration of the tetrahydrofuran solution containing poloxamer 407 is 45-55 mg / mL.

[0046] Preferably, the volume ratio of the ultra-dry tetrahydrofuran, the tetrahydrofuran solution containing the AIE material, the tetrahydrofuran solution containing poloxamer 407, and methyl orthosilicate in step S1 is 20:1:20:0.7, the concentration of the tetrahydrofuran solution containing the AIE material is 1 μM, and the concentration of the tetrahydrofuran solution containing poloxamer 407 is 50 mg / mL.

[0047] Specifically, the AIE material is AF, which is a new type of AIE material named N,N'-((Benzo[c][1,2,5]thiadiazole-4,7-diylbis(thiophene-5,2-diyl))bis(4,1-phenyl))bis(N-phenylnaphthalen-1-amine), abbreviated as AF. The structure of AF is: .

[0048] Specifically, the synthetic route of AF is as follows: Specifically, the preparation method of AF comprises the following steps: (1) Dissolve (4-(naphthalen-1-yl(phenyl)amino)phenyl)boric acid in ethanol, add a toluene solution containing 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole and a sodium carbonate aqueous solution, and then perform deoxygenation treatment; (2) injecting a toluene solution containing tetrakis(triphenylphosphine)palladium(0) and performing deoxygenation treatment; (3) Stir vigorously at 85-95 °C for more than 24 hours under nitrogen protection, cool to room temperature, and rotary evaporate to obtain a crude product; (4) The crude product was dissolved in dichloromethane and recrystallized from n-hexane, and then purified by silica gel column chromatography to obtain the final product AF.

[0049] Specifically, the equivalent ratio of (4-(naphthalene-1-yl(phenyl)amino)phenyl)boric acid, 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole and tetrakis(triphenylphosphine)palladium(0) is (18-22):(9-11):1. Preferably, the equivalent ratio of (4-(naphthalene-1-yl(phenyl)amino)phenyl)boric acid, 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole and tetrakis(triphenylphosphine)palladium(0) is 20:10:1.

[0050] Specifically, the molar volume ratio of the (4-(naphthalene-1-yl(phenyl)amino)phenyl)boric acid to ethanol is 0.25 mmol / mL, the molar volume ratio of the 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole to toluene is 0.25 mmol / mL, the concentration of the sodium carbonate aqueous solution is 1.8-2.2 M, preferably 2 M, and the volume ratio of the toluene solution to the sodium carbonate aqueous solution is 5:(1.8-2.2), preferably 5:2.

[0051] Specifically, the deoxygenation treatment is performed as follows: vacuuming, filling with nitrogen after 10 minutes, and repeating this cycle three times.

[0052] Specifically, the eluent used for the silica gel column chromatography purification is n-hexane and dichloromethane in a volume ratio of 2:1.

[0053] Specifically, after the mixing in step S1 and the addition to the stirred deionized water in step S2, a uniform treatment is further performed. Specifically, the uniform treatment is performed using an ultrasonic probe of a cell disruptor with a power of 50 W for 10 minutes and an interval of 10 seconds on / 10 seconds off.

[0054] Specifically, the stirring speed of the deionized water in step S2 is 1500 rpm, and the stirring speed after the uniform treatment in step S2 is 1500 rpm, and the duration is 5 days.

[0055] Specifically, after the uniform treatment and before the stirring in step S2, the step further includes the following steps: transferring the solution into a container, covering the bottle mouth with tin foil, and using a toothpick to pierce a plurality of small holes.

[0056] Specifically, the specific operation of dialysis in step S3 is: transferring the solution obtained in S2 into a dialysis bag with a molecular weight cut-off of 40K, and dialyzing for 3 days to remove residual THF or other water-soluble small molecule impurities.

[0057] Specifically, the specific operation of filtering in step S3 is: filtering with a water-based syringe filter with a pore size of 0.45 μm, and then filtering with a water-based syringe filter with a pore size of 0.22 μm to remove large particles of impurities.

[0058] The present invention also provides a two-photon AIE nanoparticle, which is prepared by the above-mentioned preparation method.

[0059] The present invention also provides the use of the above-mentioned two-photon AIE nanoparticles in deep imaging of biological tissues.

[0060] Specifically, two-photon AIE nanoparticles, AF@SiO2-Dex NPs, exhibit excellent biocompatibility and two-photon fluorescence properties. They can be injected into the bloodstream via the tail vein and remain in the blood vessels for extended periods. Two-photon microscopy allows for fluorescence visualization of living blood vessels. Combined with the deep imaging depth of two-photon imaging, three-dimensional reconstruction of deep tissue vascular networks is possible, enabling the study of vascular morphology and density.

[0061] The following is an explanation with reference to specific embodiments: Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The materials, reagents, etc. used in the following examples are all commercially available, unless otherwise specified. Techniques not described in detail are performed according to standard methods well known to those skilled in the art.

[0062] Example 1: Preparation and characterization of AIE material AF The preparation method of AIE material AF is as follows: (4-(naphthalen-1-yl(phenyl)amino)phenyl)boronic acid (CAS: 717888-41-0, 847 mg, 2.5 mmol, 2.0 equiv) is fully dissolved in a reaction system of 10 mL of ethanol. Then, 5 mL of toluene solution containing 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole (CAS: 288071-87-4, 575 mg, 1.25 mmol, 1.0 equiv) and 2 mL of sodium carbonate aqueous solution (2 M) are added. The mixed system is deoxygenated by three consecutive vacuum-nitrogen cycles (vacuuming for 10 minutes each time and then filling with nitrogen). Subsequently, 5 mL of toluene solution containing tetrakis(triphenylphosphine)palladium(0) (142 mg, 0.125 mmol, 0.1 equiv) is injected into the reaction system, and three additional vacuum-nitrogen cycles are performed under the same conditions. The reaction system was stirred vigorously at 85°C under nitrogen for 24 hours. After cooling to room temperature, the solvent was removed by rotary evaporation. The crude product was dissolved in dichloromethane and recrystallized from n-hexane. Further purification was performed by silica gel column chromatography (eluent: n-hexane / dichloromethane = 2 / 1, v / v) to obtain AIE material AF as a black solid (yield: 33.1%).

[0063] The sampled AIE material AF was dissolved in a deuterated reagent and analyzed on a Bruker AVANCE Ⅲ 500M superconducting nuclear magnetic resonance spectrometer. 1 H-proton spectrum and 13 Characterization of C-carbon spectrum: 1H NMR(500 MHz, Chloroform-d) δ 8.07 (d, 2H, J=5.0Hz), 7.95 (d, 2H, J=1.1 Hz), 7.90 (d, 4H, J=1.2 Hz), 7.81(s, 2H), 7.78 (d,2H, J=1.0 Hz), 7.49 (m, 8H, J=3.1 Hz), 7.37 (t, 4H, J=2.2 Hz), 7.26 (d, 2H, J=1.0 Hz), 7.23 (d, 2H, J=1.1 Hz), 7.11 (d, 4H, J=1.1 Hz), 7.01 (m, 6H, J=2.1Hz). 13 C NMR (126 MHz, Chloroform- d ) δ 152.59, 148.24, 147.90, 145.58, 143.12,137.66, 135.33, 131.19, 129.25, 128.68, 128.48, 127.32, 127.15, 126.77,126.60, 126.39, 126.26, 125.60, 125.11, 124.17, 123.00, 122.59, 122.39,121.25. The AIE material AF was dissolved in mass spectrometry grade acetonitrile and characterized by mass spectrometry using a Waters Xevo G2-XS Qtof time-of-flight tandem high-resolution mass spectrometer: ESI-MS calculation for C 58 H 38 N4S3, [M + H] + 887.2337, found887.2398. Example 2: Characterization of Fluorescence Properties of AIE Material AF The optical properties of the AIE material AF were systematically characterized by UV-Vis spectrophotometry and fluorescence spectrophotometry. Figure 5 As shown, the AIE material AF dissolved in tetrahydrofuran (THF) exhibits distinct absorption and fluorescence emission peaks at 530 nm and 715 nm, respectively, exhibiting near-infrared (NIR) emission characteristics and a Stokes shift of 195 nm. This significant spectral shift is attributed to the intramolecular charge transfer effect (ICT), which can effectively reduce self-quenching and lower biological autofluorescence background, facilitating bioimaging applications.

[0064] like Figure 6As shown, the fluorescence emission of the AIE material AF in pure acetonitrile (100%) is weak. As the water content increases from 0% to 20%, the fluorescence intensity shows a slight downward trend. This initial fluorescence decrease can be attributed to the twisted intramolecular charge transfer (TICT) effect promoted by the increased solvent polarity. However, when the water content exceeds 20%, the fluorescence intensity increases significantly, confirming that the material AF exhibits a significant focusing-induced emission (AIE) effect in its aggregated state.

[0065] The structural analysis of AIE material AF is as follows Figure 7 As shown, the thiophene-benzothiadiazole unit forms large dihedral angles of approximately 73.25°, 63.37°, and 83.82° with the adjacent aromatic benzene rings, respectively. This twisted molecular conformation effectively suppresses the π-π stacking interaction in the aggregated state. The aromatic amine group acts as a molecular rotor. During the aggregation process, it restricts intramolecular motion while retaining the intramolecular charge transfer (ICT) effect. This dual action mechanism can not only suppress the non-radiative decay pathway but also maintain the radiative transition, ultimately achieving fluorescence enhancement. In summary, the AIE material AF has great potential in the development of long-wavelength emitting fluorescent nanoparticles due to its near-infrared emission, large Stokes shift, and AIE properties.

[0066] Example 3: Preparation and characterization of nanoparticles The preparation methods of nanoparticles AF@F127 NPs, nanoparticles AF@SiO2 NPs, and nanoparticles AF@SiO2-Dex NPs are as follows: 1) To a 5 mL centrifuge tube, add ultra-dry tetrahydrofuran (THF, 1 mL), a THF solution containing the AIE material AF (50 μL, 1 μM), a THF solution containing Pluronic F127 (1 mL, 50 mg / mL), and methyl orthosilicate (TMOS, 0-100 μL). The mixture was homogenized using the ultrasonic probe of a cell disruptor (50 W, 10 min, 10 s on / 10 s off).

[0067] When 0 μL of TMOS was added, micellar nanoparticles (AF@F127 NPs) were prepared for control studies. Subsequent studies revealed that when 35 μL of TMOS was added, representing a 1.75% TMOS concentration in the 2 mL THF system, the resulting AF@SiO2 NPs exhibited optimal performance. These nanoparticles were used in subsequent testing and applications.

[0068] 2) Add 10 mL of deionized water to a 50 mL round-bottom flask. While stirring at 1500 rpm, add the tetrahydrofuran mixture dropwise in 50 μL increments. Transfer the resulting water / THF mixture to a 5 mL centrifuge tube and homogenize again using the ultrasonic probe of a cell disruptor (power: 50 W, 10 minutes, 10 seconds on / 10 seconds off). Transfer the solution back to the round-bottom flask, cover the flask with aluminum foil, poke several small holes with a toothpick, and stir at 1500 rpm for 5 days.

[0069] 3) The solution was transferred to a dialysis bag with a molecular weight cutoff of 40 K and dialyzed for 3 days to remove residual THF or other water-soluble small molecule impurities. The aqueous solution was then filtered through water filters with pore sizes of 0.45 μm and 0.22 μm to remove large particulate impurities, obtaining an aqueous solution of purified two-photon AIE nanoparticles AF@SiO2NPs.

[0070] The preparation method of nanoparticles AF@SiO2-Dex NPs further includes step 4) after step 3): 4) To a 10 mL aqueous solution of AF@SiO2 NPs, 10 mL of 1 M hydrochloric acid and 150 μL of aminopropyltriethoxysilane (APTES) were added while stirring. After stirring for 24 hours, the solution was centrifuged at 5000 rpm for 20 minutes using an ultrafiltration centrifuge tube (MWCO 100 kD). The solution was resuspended in PBS and ultrafiltered again. The nanoparticles were resuspended in a small amount of PBS and added to 10 mL of PBS containing N-hydroxysuccinimide activated ester (NHS)-modified dextran (MWCO 1.5K, 5 mM). The mixture was stirred in an ice bath for 12 hours. The solution was then centrifuged at 5000 rpm for 20 minutes using an ultrafiltration centrifuge tube (MWCO 100 kD), resuspended in ultrapure water, and ultrafiltered again to obtain purified two-photon AIE nanoparticles, AF@SiO2-Dex NPs.

[0071] NMR characterization of nanoparticles: freeze-dry the sample to remove water, resuspend it in D2O as a deuterated reagent, and then perform NMR hydrogen spectrum detection. Figure 8As shown in the figure, the NMR signals of AF@F127 NPs mainly come from poloxamer 407, whose block structure is PEO-PPO-PEO, among which 3.72 ppm is the characteristic peak of PEO, and 3.58 ppm and 1.19 ppm are from PPO; the NMR hydrogen spectrum signals of AF@SiO2NPs are similar to those of AF@F127 NPs, and the characteristic peaks are all from PEO (3.72 ppm) and PPO (3.58 ppm and 1.19 ppm); the signals of N-hydroxysuccinimide activated ester (NHS) modified dextran (Dextran-NHS) at 5.00, 4.00, 3.93, 3.78, 3.74, 3.60, and 3.54 ppm are from glucose monomers, and the characteristic peak at 2.74 ppm is from the NHS group. In the hydrogen spectrum of AF@SiO2-Dex NPs, characteristic peaks of poloxamer 407 (3.72, 3.60, 1.19 ppm) and dextran (5.00, 4.02, 3.92, 3.78, 3.74, 3.60, 3.54 ppm) were observed, and the original characteristic peak belonging to the NHS group (2.74 ppm) disappeared, suggesting that dextran was successfully modified on the surface of the silica layer of the nanoparticles through amidation coupling.

[0072] Example 4: Effect of Silica Layer Encapsulation on Nanoparticle Properties The physical properties of micellar AIE nanoparticles AF@F127 NPs without silica layer and nanoparticles AF@SiO2 NPs with silica layer were compared. Figure 9 As shown in Figure 2, AF@F127 NPs and AF@SiO2 NPs have similar absorption peak wavelengths (520 nm and 523 nm). Figure 10 As shown in the figure, the emission peak wavelengths of the two are similar (710 nm and 703 nm). It is worth noting that under the same AF concentration, the fluorescence emission intensity of AF@SiO2NPs is significantly enhanced by 3.34 times compared with AF@F127NPs. Figure 11 As shown in Figure 2, dynamic light scattering (DLS) tests showed that AF@F127 NPs and AF@SiO2NPs had similar hydrodynamic particle sizes of 25.91 ± 3.77 nm and 28.87 ± 2.14 nm, respectively, suggesting that the introduction of the silica layer did not significantly affect the hydrodynamic properties of the nanoparticles. Transmission electron microscopy (TEM) images showed that the micellar AF@F127 NPs were amorphous ( Figure 12 ); while AF@F127 NPs showed a distinct concentric morphology, with a silica shell thickness of approximately 8.31 ± 0.74 nm ( Figure 13 ).like Figure 14As shown in the figure, the Zeta potential of AF@F127 NPs is smaller, which is -0.54±0.09 mV, due to the electroneutrality of Pluronic F127. However, due to the protonation of silanol groups on the surface of the silica layer of AF@SiO2NPs, it has an increased Zeta potential of -24.22±0.51 mV.

[0073] Example 5: Optimization of the fluorescence properties of AIE nanoparticles enhanced by silica encapsulation During the preparation of nanoparticles, different amounts (0-100 μL) of TMOS were introduced using the controlled variable method to optimize the fluorescence properties of the nanoparticles. Figure 15 As shown in Figure 2, when the amount of TMOS added was 0-35 μL, the AF@SiO2NPs solution remained clear, and DLS measurements showed that the hydrodynamic size of the nanoparticles was approximately 25 nm, indicating that the silica shell encapsulation of the AIE hydrophobic core did not significantly change its hydrodynamic properties. When the amount of TMOS added increased to 40-60 μL, the AF@SiO2NPs solution gradually became turbid, and the DLS value increased significantly, suggesting that the nanoparticles tended to bind to the hydrolyzed TMOS, resulting in aggregation and eventual precipitation ( Figure 16 ). TEM images show that as the TMOS concentration increases, the silicon dioxide layer thickens and obvious particle aggregation occurs ( Figure 17 When the amount of TMOS added reached 70-100 μL, a pink precipitate appeared in the solution due to the hydrolysis of excess TMOS, and the supernatant became colorless, indicating that no AIE nanoparticles were dispersed in the supernatant. Figure 18 The effect of silica encapsulation on the fluorescence properties of AF@SiO2 NPs is shown. The control group consisted of micellar AF@F127 NPs without silica encapsulation (i.e., with TMOS added = 0 μL). Within the range of 0-35 μL (0-1.75%) TMOS, the fluorescence enhancement of the AF@SiO2 NPs was dose-dependent (reaching a maximum of 3.13-fold at 35 μL TMOS). This enhancement is attributed to the centripetal compression of the silica shell on the AIE hydrophobic core, effectively enhancing the AIE effect. When the amount of TMOS added was increased to 40-100 μL, despite significant particle aggregation and precipitation, the overall fluorescence intensity of the solution remained relatively stable, suggesting that the outward-growing silica shell, after reaching a certain thickness, can no longer exert further centripetal compression on the encapsulated AIE material. Therefore, it was confirmed that when the volume of TMOS added was 35 μL, that is, when TMOS accounted for 1.75% in the above 2 mL THF system, the obtained nanoparticles AF@SiO2NPs had better hydrodynamic and fluorescence properties.

[0074] Example 6: Effect of silica encapsulation on the two-photon performance of nanoparticles The above optimization strategy (TMOS was added in 35 μL, accounting for 1.75% in the system) was used to prepare nanoparticles AF@SiO2NPs, and the two-photon performance was compared with that of nanoparticles AF@F127NPs without silica encapsulation. Figure 19 As shown in the figure, the absolute fluorescence quantum yield (QY = the ratio of the number of emitted photons to the number of absorbed photons) was measured by an Edinburgh FLS1000 fluorescence spectrophotometer. The fluorescence quantum yields of AF@F127 NPs and AF@SiO2 NPs were QY1 = 12.23 ± 1.95% and QY2 = 48.76 ± 3.09%, respectively. The latter was 3.99 times that of the former, indicating that silica encapsulation effectively improved the fluorescence properties of the nanoparticles.

[0075] To investigate the effect of silica encapsulation on the nonlinear optical properties of AIE nanoparticles, e.g. Figure 20 As shown, a Leica SP8 DIVE multiphoton confocal microscope equipped with a Coherent Chameleon two-photon laser system was used to illuminate a methanol sample containing AF@F127 NPs or AF@SiO2NPs in a capillary sample tube, with rhodamine B (RB) as a reference. Under femtosecond laser excitation in the wavelength range of 820-1080 nm, fluorescence images of the samples in the glass capillary tube were acquired and the fluorescence intensity values ​​were calculated. The fluorescence intensity of the nanoparticles AF@F127 NPs and AF@SiO2NPs showed a linear relationship with the logarithm of the excitation power, with R²=0.9958 and R²=0.9954, respectively, indicating that they have two-photon optical properties ( Figure 21 ).like Figure 22 As shown, within the 820-1080nm near-infrared spectral range, AF@SiO2NPs exhibit significantly enhanced two-photon absorption cross sections (2PACS), with a particularly characteristic peak at 1060nm. Its absorption cross section is 256.56±14.72 GM, a 4.37-fold enhancement compared to AF@F127NPs (49.63±8.48 GM). This suggests that AF@SiO2NPs can achieve more efficient excitation at significantly reduced laser power. The reduced excitation power requirement not only helps reduce photobleaching of the nanoparticles, but also effectively reduces phototoxic damage to biological tissues, thereby significantly improving the biocompatibility of the system in long-term imaging applications.

[0076] Example 7: Characterization of the properties of dextran-modified nanoparticles AF@SiO2-Dex NPs Furthermore, dextran (1.5K) was modified on the surface of the silica layer of AF@SiO2NPs to enhance the biocompatibility of the nanoparticles. Figure 23As shown in Figure 2, TEM results suggest that AF@SiO2-Dex NPs have a core-shell structure similar to that of AF@SiO2NPs. Figure 24 As shown in the figure, the absorption and emission peaks of AF@SiO2-Dex NPs are 525 nm and 706 nm, respectively. The fluorescence quantum efficiency QY3 was measured to be 45.90±2.39%, and the 2PACS at 1060 nm was 256.08±10.97 GM. The fluorescence performance was comparable to that of AF@SiO2NPs, indicating that the dextran modification did not affect the fluorescence performance of the nanoparticles. Figure 25 As shown in Figure 2, the hydrodynamic particle size of AF@SiO2-DexNPs measured by DLS was 33.96 ± 3.03 nm. Figure 26 As shown in the figure, the Zeta potential of AF@SiO2-Dex NPs was measured to be -0.73±0.12 mV. The absolute value of the Zeta potential was significantly lower than that of AF@SiO2NPs (-24.22±0.51 mV), indicating that it can better avoid protein adsorption and further improve biocompatibility.

[0077] Example 8: Comparison of Stability of Nanoparticles like Figure 27 As shown in the figure, equal amounts (10 μM) of aqueous solutions of AF@F127 NPs, AF@SiO2 NPs, and AF@SiO2-Dex NPs were allowed to stand for one month. The supernatants were collected every five days, and the fluorescence intensity was measured using a fluorescence spectrophotometer. The fluorescence of the supernatant in the AF@F127 NPs group was observed to gradually decrease compared to day 0, and a small purple precipitate was observed to gradually appear in the solution. This is speculated to be due to the collapse of the micellar nanoparticles AF@F127 NPs, the precipitation of the AIE material AF, and the resulting decrease in the fluorescence intensity of the supernatant. The fluorescence intensity of the samples in the AF@SiO2 NPs and AF@SiO2-Dex NPs groups remained essentially unchanged, demonstrating improved stability.

[0078] Example 9: Cytotoxicity of Nanoparticles AF@SiO2-Dex NPs In order to deeply evaluate the biocompatibility of the nanoparticles AF@SiO2-Dex NPs after combining the above-mentioned multiple materials, the classic immortalized HeLa cells and HUVEC cells that are crucial for angiogenesis were selected for experiments. Figure 28As shown in the figure, the two cell types were treated with different concentrations (0-50 μM) of AF@SiO2-Dex NPs for 48 hours, and the cell viability was measured using a CCK8 assay. The results showed that both cell types maintained extremely high viability (>95%) after treatment with AF@SiO2-Dex NPs, indicating that AF@SiO2-Dex NPs have low cytotoxicity and high biocompatibility.

[0079] Example 10: In vivo safety assessment of nanoparticles AF@SiO2-Dex NPs To evaluate the in vivo safety of AF@SiO2-Dex NPs, female C57BL / 6 mice (6-7 weeks old) were intravenously injected with AF@SiO2-Dex NPs (100 μL, dose groups: 0, 5, 50 μM, respectively) on days 1 and 7 of a 14-day experimental cycle. Figure 29 As shown in the figure, the weight gain trend of mice in each group was consistent with that of the control group (0 μM). Continuous behavioral monitoring revealed no abnormalities in the feeding behavior, motor activity, and excretion function of the experimental animals. The mice were killed on the 14th day, and the main organs were obtained and weighed. The organ coefficient (organ / body weight ratio) was calculated. The results showed that there was no significant difference between the experimental groups and the control group ( Figure 30 ), Figure 31 On the 14th day, blood was collected from mice and key hematological parameters were tested. The results showed that white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean hemoglobin content (MCH), mean hemoglobin concentration (MCHC) and red blood cell distribution width variation coefficient (RDW-CV) were all within the physiological range, confirming normal hematopoietic function and the absence of hepatotoxicity and renal toxicity. Pathological evaluation of fixed sections of important organs (heart, liver, spleen, lung, kidney, uterus) showed normal tissue structure, with no inflammatory infiltration or pathological changes ( Figure 32 The above comprehensive evidence confirms that the nanoparticles AF@SiO2-Dex NPs have good biocompatibility and potential for biomedical applications.

[0080] Example 11: Visualization of Mouse Uterine Blood Vessels AF@SiO2-Dex NPs possess high biocompatibility, high fluorescence quantum efficiency, and high two-photon absorption cross-section, making them promising for bioimaging applications. Here, to investigate the dynamics of uterine vessels in early pregnancy and non-pregnant uteri, AF@SiO2-Dex NPs were used to perform fluorescence visualization of the mouse uterine vascular network.

[0081] First, the mouse uterus was exposed, including anesthesia, abdominal hair removal, and limb fixation. After creating a 1 cm incision in the mouse abdomen, the uterine tissue was gently exteriorized. After the nanoparticles AF@SiO2-Dex NPs were injected into the mouse tail vein, imaging was performed using a 25x objective lens of a two-photon microscope. Based on the characteristics of the uterine serosal layer and myometrium being rich in collagen fibers, a two-photon microscope was used to excite the uterine tissue at 960 nm, and the typical bundle structure of collagen fibers was clearly displayed using the second harmonic generation (SHG) imaging mode ( Figure 33 The 960 nm excitation light can also effectively excite the near-infrared fluorescence of the nanoparticles AF@SiO2-DexNPs, enabling visualization of the vascular network ( Figure 33 b) in the above example.

[0082] It is noteworthy that the SHG signal intensity in the vascular lumen is significantly attenuated compared to the surrounding matrix tissue. This phenomenon is due to the inherent optical properties of blood: as a homogeneous liquid phase composed mainly of water and cellular components, blood lacks the collagen matrix structure required for efficient SHG emission. Therefore, the vascular outline precisely occupies the branched low-intensity area in the SHG image ( Figure 33 The above spatial complementary results verify the ability of SHG imaging and two-photon imaging of nanoparticles AF@SiO2-Dex NPs to analyze the spatial relationship between the vascular system and the extravascular matrix.

[0083] As the imaging depth increases, Figure 33 As shown in (d), SHG imaging within the endometrial layer shows multiple cavity-like structures, which are endometrial glands. Figure 33 e and Figure 33 Figure f shows that AF@SiO2-Dex NPs can image the capillary network (4-10 μm in diameter) around the gland. Figure 33 g) and AF@SiO2-Dex NPs ( Figure 33 h) channel in the z-axis layer scanning, achieving three-dimensional reconstruction of uterine tissue at a depth of more than 300 μm ( Figure 33 The nanoparticles AF@SiO2-DexNPs exhibited excellent optical penetration properties, enabling two-photon depth imaging, providing a comprehensive research tool for analyzing the spatial relationship of uterine heterogeneity.

[0084] Example 12: Nanoparticles AF@SiO2-Dex NPs used to study vascular permeability Fluorescently labeled dextran (e.g., FITC-dextran) is widely used in vascular permeability studies due to its good biodegradability and low immunogenicity. Here, mice were injected with nanoparticles AF@SiO2-Dex NPs and FITC-dextran through the tail vein, and then two-photon imaging of the mouse uterus was performed. In the early stages of injection, collagen fibers were visualized with SHG imaging as the background ( Figure 34 (a) Nanoparticles AF@SiO2-Dex NPs and FITC-dextran are both confined to the blood vessels ( Figure 34 b- Figure 34 Colocalization analysis showed a significant spatial correlation between the two probes (Pearson coefficient R = 0.96); linear scanning fluorescence intensity spectra further confirmed that the two had overlapping distribution patterns ( Figure 35 ).

[0085] Figure 36 Shown, approximately 60 minutes after injection, with collagen fibers as the background ( Figure 36 (a) The nanoparticles AF@SiO2-Dex NPs still maintain continuous vascular retention properties and stable fluorescence intensity, indicating their potential as real-time fluorescent angiography agents ( Figure 36 In contrast, the FITC-dextran signal gradually decays within the blood vessels, accompanied by significant extravasation into the perivascular tissues ( Figure 36 At this time, the colocalization coefficient was significantly reduced to R = 0.21, and the fluorescence intensity spectrum showed that the distribution patterns of the two were significantly differentiated ( Figure 37 The results demonstrate the potential of using AF@SiO2-Dex NPs combined with FITC-dextran to study uterine vascular transport and diffusion mechanisms.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing two-photon AIE nanoparticles, characterized in that: The following steps are involved: S1. The tetrahydrofuran, the tetrahydrofuran solution containing the AIE material, the tetrahydrofuran solution containing poloxamer 407 and methyl orthosilicate are mixed to obtain a mixed solution; S2 S1 obtained by adding the mixture dropwise to the stirring deionized water; S3. Dialysis and filtration were performed to obtain purified two-photon AIE nanoparticles AF@SiO2 NPs.

2. The preparation method according to claim 1, wherein After step S3, step S4 is also included: adding the two-photon AIE nanoparticles AF@SiO2 NPs to hydrochloric acid and aminopropyltriethoxysilane while stirring, ultrafiltering twice after stirring, adding PBS to resuspend, adding to PBS containing N-hydroxysuccinimide activated ester-modified dextran, stirring in an ice bath, ultrafiltering, adding ultrapure water to resuspend, and ultrafiltering again to obtain purified two-photon AIE nanoparticles AF@SiO2-DexNPs.

3. The preparation method according to claim 2, wherein In step S4, the volume ratio of the two-photon AIE nanoparticles AF@SiO2 NPs, the hydrochloric acid, the aminopropyltriethoxysilane, and the PBS containing the dextran modified with N-hydroxysuccinimide activated ester is (180-220):(180-220):3:(180-220), the concentration of the hydrochloric acid is 0.9-1.1 M, and the concentration of the dextran is 4.5-5.5 mM; And / or, the specific operation of the ultrafiltration is: using an ultrafiltration centrifuge tube with a molecular weight cut-off of 100 kD and centrifuging at 5000 rpm for 18-22 minutes; and / or, adding hydrochloric acid and aminopropyltriethoxysilane, stirring for 22-26 hours, and stirring in an ice-water bath for 10.8-13.2 hours.

4. The preparation method according to claim 1, wherein In step S1, the volume ratio of tetrahydrofuran, the tetrahydrofuran solution containing the AIE material, the tetrahydrofuran solution containing poloxamer 407, and methyl orthosilicate is (18-22):1:(18-22):(0.7-0.8). The concentration of the tetrahydrofuran solution containing the AIE material is 0.9-1.1 μM, and the concentration of the tetrahydrofuran solution containing poloxamer 407 is 45-55 mg / mL.

5. The preparation method according to claim 1, wherein The AIE material is AF, and the structure of AF is: , the preparation method of AF comprises the following steps: (1) (4-(naphthalen-1-yl(phenyl)amino)phenyl)boric acid was fully dissolved in ethanol, and a toluene solution containing 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole and a sodium carbonate aqueous solution were added, followed by deoxygenation treatment; (2) injecting a toluene solution containing tetrakis(triphenylphosphine)palladium(0) and performing deoxygenation treatment; (3) Stir vigorously at 85-95°C under nitrogen protection for more than 24 hours, cool to room temperature, and rotary evaporate to obtain the crude product; (4) The crude product was dissolved in dichloromethane and recrystallized from n-hexane, and then purified by silica gel column chromatography to obtain the final product AF.

6. The preparation method according to claim 5, wherein The equivalent ratio of (4-(naphthalen-1-yl(phenyl)amino)phenyl)boric acid, 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole and tetrakis(triphenylphosphine)palladium(0) is (18-22):(9-11):1; The molar volume ratio of the (4-(naphthalen-1-yl(phenyl)amino)phenyl)boric acid to ethanol is 0.25 mmol / mL, the molar volume ratio of the 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole to toluene is 0.25 mmol / mL, the concentration of the sodium carbonate aqueous solution is 1.8-2.2 M, and the volume ratio of the toluene solution to the sodium carbonate aqueous solution is 5:(1.8-2.2).

7. The preparation method according to claim 5, wherein The specific operation of the deoxygenation treatment is: vacuuming, filling with nitrogen after 10 minutes, and repeating this cycle 3 times; And / or, the eluent used for the silica gel column chromatography purification is n-hexane and dichloromethane in a volume ratio of 2:

1.

8. The preparation method according to claim 1, wherein After the mixing in step S1 and the addition to the stirred deionized water in step S2, a homogenization treatment is further performed. The specific operation of the homogenization treatment is as follows: the homogenization treatment is performed using an ultrasonic probe of a cell disruptor at a power of 50 W for 10 minutes with an interval of 10 seconds on / 10 seconds off; and / or, the stirring speed of the deionized water in step S2 is 1500 rpm, and the stirring speed after the uniform treatment in step S2 is 1500 rpm for 5 days; And / or, after the uniform treatment in step S2 and before the stirring, the step further includes: transferring the solution into a container, covering the bottle opening with tin foil and punching a plurality of small holes in the foil; And / or, the specific operation of dialysis in step S3 is: transferring the solution obtained in S2 into a dialysis bag with a molecular weight cut-off of 40K and dialysis for 3 days; And / or, the specific operation of filtering in step S3 is: filtering using a water-based syringe filter with a pore size of 0.45 μm, and then filtering using a water-based syringe filter with a pore size of 0.22 μm.

9. A two-photon AIE nanoparticle, characterized in that: The method is as described in any one of claims 1 to 8.

10. Use of the two-photon AIE nanoparticles according to claim 9 in depth imaging of biological tissues.

Images