A two-photon type AIE nanoparticle, a preparation method and application thereof

Abstract

This invention discloses a two-photon AIE nanoparticle, the preparation method of which includes the following steps: S1. Mixing tetrahydrofuran, a tetrahydrofuran solution containing AIE material, a tetrahydrofuran solution containing poloxamer 407, and methyl orthosilicate, and homogenizing the mixture to obtain a homogenized solution; S2. Adding the mixture obtained in S1 dropwise to deionized water under stirring, then homogenizing the mixture again, and then stirring; S3. Dialyzing and then filtering to obtain purified two-photon AIE nanoparticles; S4. Modifying the surface of silica with amino groups and then modifying it with dextran to obtain dextran-modified two-photon AIE nanoparticles. The preparation method disclosed in this invention has the advantages of simple preparation and low cost. 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] This invention relates to the field of biochemistry, specifically to a two-photon AIE nanoparticle, its preparation method, and its applications. Background Technology

[0002] Aggregation-induced emission (AIE) is a newly emerging fluorescence generation technique. Fluorescent materials with AIE properties do not emit light or only exhibit weak fluorescence in dilute solutions, but show significantly enhanced fluorescence in high-concentration or solid aggregated states. Current research on the mechanism of the AIE phenomenon suggests that AIE material molecules generally possess a unique propeller structure. In the dispersed state, after excitation, the propeller structure can rotate freely to consume radiative energy; in the aggregated state, the intermolecular motion is restricted, and after excitation, it can greatly avoid non-radiative decay, exhibiting enhanced fluorescence.

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

[0004] The classic strategy for preparing AIE NPs leverages the lipophilic nature of AIE materials by utilizing amphiphilic materials (such as poloxamer 407, or Pluronic F127) for self-assembly encapsulation, resulting in nanomicelles encapsulating AIE materials. The steps are briefly described as follows: Pluronic F127 and AIE materials are mixed in a volatile organic solvent (such as tetrahydrofuran) and then added dropwise to water, allowing Pluronic F127 to self-assemble into nanomicelles. During the formation of nanomicelles, the lipophilic AIE material is encapsulated within the hydrophobic core of the Pluronic F127 nanomicelles, with the outer edge of the micelles consisting of hydrophilic long chains, thus yielding water-soluble AIE NPs. The solution is continuously stirred to evaporate and remove the organic solvent, and further purification is achieved through 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) emitted near-infrared (NIR) fluorescence upon excitation and were used for two-photon depth imaging of mouse uterine vessels (DOI: 10.1038 / s41467-023-40728-6). Xiaoming Yu et al. developed AIE NPs (2FT-oCB dots) using a similar strategy and used two-photon microscopy to achieve uterine cavity imaging and uterine angiography, which has both biosafety and clinical applicability (DOI: 10.1016 / j.nantod.2021.101235).

[0005] As shown above, AIE materials can be prepared by encapsulating them with amphiphilic materials to create AIE NPs. This strategy is convenient, simple, and effective, and has significant application value in biofluorescence imaging. However, the AIENPs prepared by the above-mentioned classical strategy have a nanomicelle structure. In a biological environment, when the micelle concentration is below the critical micelle concentration (CMC), dissociation easily occurs, leading to AIE material leakage, fluorescence background interference, and even toxic side effects. Furthermore, when using two-photon technology for deep tissue imaging, AIE NPs are required to have good two-photon performance. Currently, the two-photon performance (such as fluorescence quantum efficiency and two-photon absorption cross-section) of micelle-type AIE NPs needs further improvement.

[0006] Therefore, research on how to improve the stability, fluorescence properties, and biocompatibility of AIE NPs is of significant scientific importance and practical value. Summary of the Invention

[0007] To address the aforementioned issues, this invention provides a two-photon AIE nanoparticle, its preparation method, and its application, for preparing nanoparticles with high stability, high safety, and high two-photon performance for two-photon imaging of deep biological tissues.

[0008] This invention provides a method for preparing two-photon AIE nanoparticles, comprising the following steps:

[0009] S1. Tetrahydrofuran, a tetrahydrofuran solution containing AIE material, a tetrahydrofuran solution containing poloxamer 407 (Pluronic F127), and methyl orthosilicate are mixed to obtain a mixture.

[0010] S2. Add the mixture obtained in S1 dropwise to the deionized water while stirring;

[0011] S3. Dialysis and filtration were performed to obtain purified two-photon AIE nanoparticles AF@SiO2NPs.

[0012] Furthermore, after step S3, step S4 is also included: the two-photon AIE nanoparticles AF@SiO2NPs are added to hydrochloric acid and aminopropyltriethoxysilane under stirring, stirred and then ultrafiltered twice, resuspended in PBS, added to PBS containing dextran modified with N-hydroxysuccinimide activated ester, stirred in an ice bath, ultrafiltered, resuspended in ultrapure water, and ultrafiltered again to obtain purified two-photon AIE nanoparticles AF@SiO2-Dex NPs.

[0013] 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.

[0014] Furthermore, the specific operation of the ultrafiltration is as follows: using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kD, centrifuge at 5000 rpm for 18-22 minutes.

[0015] Further, hydrochloric acid and aminopropyltriethoxysilane were added, and the mixture was stirred for 22-26 h, followed by stirring in an ice-water bath for 10.8-13.2 h.

[0016] Furthermore, in step S1, the volume ratio of tetrahydrofuran, the tetrahydrofuran solution containing 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 AIE material is 0.9-1.1 μM, and the concentration of the tetrahydrofuran solution containing poloxamer 407 is 45-55 mg / mL.

[0017] 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-phenylnaphthyl-1-amine), and the structure of the AF is:

[0018] .

[0019] Furthermore, the synthesis route of the AF is as follows:

[0020]

[0021] Furthermore, the preparation method of the AF includes the following steps:

[0022] (1) Dissolve (4-(naphthyl-1-yl(phenyl)amino)phenyl)boronic acid completely in ethanol, add toluene solution containing 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole and sodium carbonate aqueous solution, and then perform deoxygenation treatment;

[0023] (2) Inject a toluene solution containing tetra(triphenylphosphine)palladium(0) and perform deoxygenation treatment;

[0024] (3) Stir vigorously at 85-95 °C for more than 24 hours under nitrogen protection, cool to room temperature, and then rotary evaporate to obtain crude product;

[0025] (4) The crude product was dissolved in dichloromethane and recrystallized in n-hexane, and then purified by silica gel column chromatography to obtain the final product AF.

[0026] Furthermore, the equivalent ratio of (4-(naphthyl-1-yl(phenyl)amino)phenyl)boronic acid, 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole and tetra(triphenylphosphine)palladium(0) is (18-22):(9-11):1.

[0027] Furthermore, the molar volume ratio of (4-(naphthyl-1-yl(phenyl)amino)phenyl)boronic acid to ethanol is 0.25 mmol / mL, the molar volume ratio of 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).

[0028] Furthermore, the specific operation of the deoxygenation treatment is as follows: vacuuming, filling with nitrogen after 10 minutes, and repeating this cycle 3 times.

[0029] Furthermore, the eluent used in the silica gel column chromatography purification is a mixture of n-hexane and dichloromethane in a volume ratio of 2:1.

[0030] Furthermore, after mixing in step S1 and adding to the deionized water in step S2, a homogenization process is also included. Specifically, the homogenization process is performed using the ultrasonic probe of the cell disruptor at a power of 50 W for 10 minutes, with an interval of 10 seconds on and 10 seconds off.

[0031] Furthermore, in step S2, the stirring speed of the deionized water is 1500 rpm, and the stirring speed after uniform treatment in step S2 is 1500 rpm for 5 days.

[0032] Furthermore, before stirring after uniform treatment in step S2, the method further includes the following steps: transferring the solution into a container, covering the bottle opening with aluminum foil, and using a toothpick to pierce multiple small holes.

[0033] Furthermore, the specific operation of dialysis in step S3 is as follows: the solution obtained in S2 is transferred to a dialysis bag with a molecular weight cutoff of 40K, and dialysis is performed for 3 days.

[0034] Furthermore, the specific filtration operation in step S3 is as follows: use a water-based syringe filter with a pore size of 0.45μm for filtration, and then use a water-based syringe filter with a pore size of 0.22μm for filtration.

[0035] The present invention also provides a two-photon AIE nanoparticle, which is prepared by the preparation method described above.

[0036] This invention also provides the application of the two-photon AIE nanoparticles described above in depth imaging of biological tissues.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] This invention provides a method for preparing two-photon AIE nanoparticles AF@SiO2NPs and AF@SiO2-Dex NPs, which has the advantages of simple preparation and low cost. The prepared nanoparticles have the following beneficial effects:

[0039] (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 micelles containing AIE. The silica layer of the obtained nanoparticles AF@SiO2NPs and AF@SiO2-Dex NPs can effectively inhibit the dissociation of micelles, which plays a role in enhancing the stability of nanoparticles and inhibiting the leakage of AIE materials. In the long-term (1 month) static test, they have better stability than nanoparticles AF@F127 NPs prepared based on the classic strategy (using only poloxamer 407 without forming a silica layer).

[0040] (2) Enhanced biocompatibility of nanoparticles: The silica layer of AF@SiO2NPs can be easily modified by hydrolysis of silicon-based materials to achieve engineered modification; further surface modification of AF@SiO2NPs with dextran makes the nanoparticles electrically neutral, 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 cell experiments and animal experiments, and had no significant effect on cell morphology and survival rate; in the biocompatibility experiment of mouse tail vein injection of AF@SiO2-Dex NPs for 2 weeks, it was found that the nanoparticles did not cause significant developmental inhibition, and had no significant effect on blood and organ function.

[0041] (3) Enhanced two-photon fluorescence performance of nanoparticles: By constructing a silica layer on the outer edge of the AIE hydrophobic core of micelle nanoparticles, the hydrophobic core of the AIE material AF of the obtained nanoparticles AF@SiO2NPs and AF@SiO2-Dex NPs is subjected to centripetal compression by the silica layer, which greatly enhances the aggregation-induced emission effect of the AIE material AF. Compared with nanoparticles AF@F127NPs prepared based on the classical strategy (using only poloxamer 407 without generating a silica layer), AF@SiO2NPs and AF@SiO2-Dex NPs showed significant improvements in two-photon fluorescence performance, including fluorescence quantum efficiency and two-photon absorption cross-section: the fluorescence quantum efficiency of AF@F127 NPs was 12.23±1.95%, while that of AF@SiO2NPs and AF@SiO2-Dex NPs were 48.76±3.09% and 45.90±2.39%, respectively; the two-photon absorption cross-section of AF@F127 NPs at the 2x absorption peak (1060 nm band) was 49.63±8.48 GM, while that of AF@SiO2NPs and AF@SiO2-Dex NPs were 266.56±14.72 GM and 256.08±10.97 GM, respectively. The enhanced fluorescence efficiency and two-photon absorption cross section result in better excitation efficiency of AF@SiO2NPs and AF@SiO2-Dex NPs after two-photon excitation. This is beneficial for reducing the required excitation light power in practical two-photon imaging applications, significantly reducing the phototoxicity of excitation light to living tissues, extending observation time, and improving experimental safety.

[0042] (4) Nanoparticles are suitable for two-photon vascular imaging: In two-photon imaging experiments in live mice, AF@SiO2-Dex NPs nanoparticles were injected into the bloodstream of mice via the tail vein. The vascular network of the mouse uterus was visualized using a two-photon microscope, with an imaging depth of over 500 μm. The vascular network was then reconstructed in three dimensions using optical slice sequences. Co-imaging of AF@SiO2-Dex NPs with FITC-dextran confirmed that AF@SiO2-Dex NPs could remain in blood vessels for a longer period. The modification of FITC-dextran reduced the immunogenicity of the nanoparticles and effectively prolonged their residence time in the bloodstream. Simultaneous imaging of blood vessels with AF@SiO2-Dex NPs nanoparticles showed the diffusion of FITC-dextran in tissues, suggesting its potential for studying vascular permeability. These results indicate that the two-photon AIE nanoparticles AF@SiO2-Dex NPs have good two-photon deep tissue imaging performance and have good application potential in biofluorescence imaging and labeling. Attached Figure Description

[0043] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 Here is a structural diagram of AF@SiO2NPs nanoparticles;

[0045] Figure 2 The synthesis route diagram for AF@SiO2NPs nanoparticles;

[0046] Figure 3 The structure diagram of AF@SiO2-Dex NPs is shown.

[0047] Figure 4 The synthesis route diagram for AF@SiO2-Dex NPs nanoparticles;

[0048] Figure 5 The fluorescence properties of the AIE material AF are shown in the diagram.

[0049] Figure 6 This is a graph showing the aggregation-induced emission properties of the AIE material AF.

[0050] Figure 7 The optimized geometric molecular structure and dihedral angle distribution of AIE material AF;

[0051] Figure 8 The 1H NMR spectra of nanoparticles AF@F127 NPs, AF@SiO2 NPs, AF@SiO2-Dex NPs and Dexran-NHS are shown.

[0052] Figure 9 The UV-Vis absorption spectra of nanoparticles AF@F127 NPs and AF@SiO2 NPs are shown.

[0053] Figure 10 The fluorescence emission spectra of nanoparticles AF@F127 NPs and AF@SiO2 NPs are shown.

[0054] Figure 11 DLS particle size distribution diagram of nanoparticles AF@F127 NPs and AF@SiO2 NPs;

[0055] Figure 12 TEM morphology of AF@F127 NPs nanoparticles;

[0056] Figure 13 The TEM morphology of AF@SiO2NPs nanoparticles is shown.

[0057] Figure 14 The zeta potential distribution of nanoparticles AF@F127 NPs and AF@SiO2 NPs is shown.

[0058] Figure 15 Images obtained by taking white light and fluorescence photographs of AF@SiO2NPs nanoparticles with different amounts of TMOS;

[0059] Figure 16 DLS particle size variation diagram of AF@SiO2NPs nanoparticles with different amounts of TMOS added;

[0060] Figure 17 TEM morphology of AF@SiO2NPs nanoparticles with different amounts (40µL and 60µL) of TMOS (scale bar 50 nm).

[0061] Figure 18 Fluorescence enhancement factor diagrams of AF@SiO2NPs nanoparticles with different amounts of TMOS added;

[0062] Figure 19 The fluorescence quantum efficiency diagrams of nanoparticles AF@F127 NPs and AF@SiO2 NPs are shown.

[0063] Figure 20 Two-photon excitation images of nanoparticles AF@F127 NPs, AF@SiO2 NPs, and reference Rhodamine B (RB) loaded in capillary glass tubes (scale bar is 100 μm).

[0064] Figure 21 The logarithmic distribution of two-photon excitation power versus fluorescence intensity for nanoparticles AF@F127 NPs and AF@SiO2 NPs is shown.

[0065] Figure 22 Two-photon absorption cross sections of nanoparticles AF@F127 NPs and AF@SiO2 NPs at different wavelengths;

[0066] Figure 23 The TEM morphology of AF@SiO2-Dex NPs is shown.

[0067] Figure 24 The fluorescence properties of AF@SiO2-Dex NPs are shown in the figure.

[0068] Figure 25 The DLS particle size distribution of AF@SiO2-Dex NPs is shown.

[0069] Figure 26The zeta potential distribution of nanoparticles AF@SiO2NPs and AF@SiO2-Dex NPs is shown.

[0070] Figure 27 The fluorescence intensity of nanoparticles AF@F127 NPs, AF@SiO2 NPs and AF@SiO2-Dex NPs changes over time.

[0071] Figure 28 The results of a toxicity study on AF@SiO2-Dex NPs nanoparticles to HeLa cells and HUVEC cells;

[0072] Figure 29 Figure showing the changes in body weight of mice after the addition of different concentrations of AF@SiO2-Dex NPs nanoparticles;

[0073] Figure 30 Organ coefficients for different organs with different concentrations of AF@SiO2-Dex NPs added;

[0074] Figure 31 Blood biochemical parameters were determined by adding different concentrations of nanoparticles AF@SiO2-Dex NPs.

[0075] Figure 32 For the pathological analysis of major organs with different concentrations of AF@SiO2-Dex NPs added (scale bar is 50 μm);

[0076] Figure 33 For vascular imaging of the uterus in a live mouse using nanoparticles AF@SiO2-Dex NPs (scale bar 100 μm);

[0077] Figure 34 Fluorescence imaging of mice 10 minutes after co-injection of AF@SiO2-Dex NPs and commercial FITC dextran via tail vein (scale bar: 100 μm).

[0078] Figure 35 Linear scanning fluorescence intensity spectra of AF@SiO2-Dex NPs and commercial FITC dextran injected into the tail vein of mice 10 minutes later;

[0079] Figure 36 Imaging images of mice 60 minutes after co-injection of AF@SiO2-Dex NPs and commercial FITC dextran via tail vein (scale bar: 100 μm).

[0080] Figure 37 Linear scanning fluorescence intensity spectra of AF@SiO2-Dex NPs and commercial FITC dextran co-injected into the tail vein of mice 60 minutes later. Detailed Implementation

[0081] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The present invention will be specifically described below with reference to specific embodiments.

[0082] This invention provides a method for preparing two-photon AIE nanoparticles (AF@SiO2NPs), the structure of which is as follows: Figure 1 As shown, the AIE material AF is a newly developed AIE material with good AIE properties, emitting red fluorescence in the near-infrared 1 region after excitation. Nanoparticles AF@SiO2NPs fabricated using the AIE material AF exhibit absorption peaks (530 nm) and fluorescence emission peaks (710 nm) similar to those of the AF small molecule. The fluorescence emission peak of the AF@SiO2NPs nanoparticles is located in the near-infrared 1 region, demonstrating good penetration depth in imaging applications. Its synthesis route is as follows... Figure 2 As shown.

[0083] Specifically, the preparation method includes the following steps:

[0084] S1. Mix ultra-dry tetrahydrofuran (THF) (with a water content of less than 30 ppm), a tetrahydrofuran solution containing AIE material, a tetrahydrofuran solution containing poloxamer 407, and methyl orthosilicate (TMS), and homogenize the mixture to obtain a mixture.

[0085] S2. Add the mixture obtained in S1 dropwise to the deionized water while stirring, then homogenize it again, and then stir.

[0086] S3. Dialyze and then filter to obtain purified two-photon AIE nanoparticles (AF@SiO2NPs).

[0087] Invention Principle: Silica materials possess extremely high mechanical strength and biochemical inertness. To improve the stability of AIE nanoparticles, a silica encapsulation layer is constructed around the hydrophobic core of the AIE material encapsulated in micelles. This encapsulation of the hydrophobic core inhibits micelle degradation in the biological environment and prevents leakage of the AIE material. Furthermore, the silica encapsulation of the AIE material strengthens the centripetal pressure on the hydrophobic core, enhancing aggregation-induced emission and thus improving the two-photon fluorescence performance of the AIE nanoparticles, such as fluorescence quantum efficiency and two-photon absorption cross-section, which is beneficial for two-photon in vivo tissue imaging.

[0088] In other embodiments, other AIE materials can be selected to prepare fluorescent nanoparticles with enhanced stability and aggregation-induced emission (AIE) effect using a silica encapsulation strategy. Simultaneously, a silicon source can be introduced onto the outer surface of the silica layer with the hydrophobic core for further modification, achieving various functionalizations.

[0089] Specifically, after step S3, step S4 is included. Step S4 involves further modifying the surface of the silica core with dextran, which can further improve the biocompatibility of the nanoparticles and reduce their immunogenicity, enabling them to remain in the bloodstream for a longer period of time. 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 As shown, the synthetic route of AF@SiO2-Dex NPs is as follows: Figure 4 As shown.

[0090] Specifically, step S4 involves the following steps: The two-photon AIE nanoparticles AF@SiO2NPs are added to hydrochloric acid and aminopropyltriethoxysilane (APTES) while stirring. After stirring, the mixture undergoes two ultrafiltrations, is resuspended in PBS, and then added to PBS containing N-hydroxysuccinimide activated ester (NHS) modified dextran. The mixture is stirred in an ice bath, ultrafiltered, resuspended in ultrapure water, and ultrafiltered again to obtain purified two-photon AIE nanoparticles AF@SiO2-Dex NPs.

[0091] Specifically, the feature is that, 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), preferably 200:200:3:200; the concentration of the hydrochloric acid is 0.9-1.1M, preferably 1M; and the concentration of the dextran is 4.5-5.5 mM, preferably 5 mM.

[0092] Specifically, the molecular weight of the dextran is 1.35-1.65 K, preferably 1.5 K.

[0093] Specifically, the ultrafiltration operation is as follows: using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kD, centrifuge at 5000 rpm for 18-22 minutes, preferably 20 minutes.

[0094] Specifically, hydrochloric acid and aminopropyltriethoxysilane are added and stirred for 22-26 h, preferably 24 h, and then stirred in an ice-water bath for 10.8-13.2 h, preferably 12 h.

[0095] Specifically, surface modification of AF@SiO2-Dex NPs, such as using 3-aminopropyltriethoxysilane (APTES) to modify the outer surface of the silica layer, can prepare two-photon AIE nanoparticles with amino groups on the surface, so as to further modify other chemical groups (targeting or fluorescence) or proteins.

[0096] Specifically, in step S1, the volume ratio of ultra-dry tetrahydrofuran, tetrahydrofuran solution containing AIE material, 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 AIE material is 0.9-1.1 μM, and the concentration of the tetrahydrofuran solution containing poloxamer 407 is 45-55 mg / mL.

[0097] Preferably, the volume ratio of the ultra-dry tetrahydrofuran, the tetrahydrofuran solution containing 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 AIE material is 1 μM, and the concentration of the tetrahydrofuran solution containing poloxamer 407 is 50 mg / mL.

[0098] Specifically, the AIE material is AF, a novel AIE material named N,N'-((benzo[c][1,2,5]thiadiazole-4,7-diylbis(thiophene-5,2-diyl))bis(4,1-phenyl))bis(N-phenylnaphthyl-1-amine), abbreviated as AF. The structure of AF is as follows: .

[0099] Specifically, the synthesis route of the AF is as follows:

[0100]

[0101] Specifically, the preparation method of the AF includes the following steps:

[0102] (1) Dissolve (4-(naphthyl-1-yl(phenyl)amino)phenyl)boronic acid completely in ethanol, add toluene solution containing 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole and sodium carbonate aqueous solution, and then perform deoxygenation treatment;

[0103] (2) Inject a toluene solution containing tetra(triphenylphosphine)palladium(0) and perform deoxygenation treatment;

[0104] (3) Stir vigorously at 85-95 °C for more than 24 hours under nitrogen protection, cool to room temperature, and then rotary evaporate to obtain crude product;

[0105] (4) The crude product was dissolved in dichloromethane and recrystallized in n-hexane, and then purified by silica gel column chromatography to obtain the final product AF.

[0106] Specifically, the equivalent ratio of (4-(naphthyl-1-yl(phenyl)amino)phenyl)boronic acid, 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole and tetra(triphenylphosphine)palladium(0) is (18-22):(9-11):1. Preferably, the equivalent ratio of (4-(naphthyl-1-yl(phenyl)amino)phenyl)boronic acid, 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole and tetra(triphenylphosphine)palladium(0) is 20:10:1.

[0107] Specifically, the molar volume ratio of (4-(naphthyl-1-yl(phenyl)amino)phenyl)boronic acid to ethanol is 0.25 mmol / mL, the molar volume ratio of 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.

[0108] Specifically, the deoxygenation process involves: evacuating the vacuum, filling with nitrogen after 10 minutes, and repeating this cycle three times.

[0109] Specifically, the eluent used in the silica gel column chromatography purification is a mixture of n-hexane and dichloromethane in a volume ratio of 2:1.

[0110] Specifically, after mixing in step S1 and adding to the deionized water in step S2, a homogenization process is also included. The specific operation is as follows: homogenization is performed using the ultrasonic probe of the cell disruptor at a power of 50 W for 10 minutes, with an interval of 10 seconds on and 10 seconds off.

[0111] Specifically, in step S2, the stirring speed of the deionized water is 1500 rpm, and the stirring speed after uniform treatment in step S2 is 1500 rpm for 5 days.

[0112] Specifically, before stirring after uniform treatment in step S2, the process also includes the following steps: transferring the solution to a container, covering the bottle opening with aluminum foil, and using a toothpick to poke several small holes.

[0113] Specifically, the dialysis procedure in step S3 is as follows: the solution obtained in S2 is 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.

[0114] Specifically, the filtration process in step S3 is as follows: use a water-based syringe filter with a pore size of 0.45 μm for filtration, and then use a water-based syringe filter with a pore size of 0.22 μm for filtration to remove large particulate impurities.

[0115] The present invention also provides a two-photon AIE nanoparticle, which is prepared by the preparation method described above.

[0116] This invention also provides the application of the two-photon AIE nanoparticles described above in depth imaging of biological tissues.

[0117] Specifically, two-photon AIE nanoparticles AF@SiO2-Dex NPs exhibit good biocompatibility and two-photon fluorescence properties, and can remain in blood vessels for extended periods after being injected into the bloodstream via the tail vein. Fluorescence visualization of living blood vessels can be achieved using two-photon microscopy. Combined with the deep imaging depth advantage of two-photon imaging, three-dimensional reconstruction of deep tissue vascular networks can be performed for studying vascular morphology and density.

[0118] The following description is based on specific embodiments:

[0119] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available, and techniques not described in detail were performed according to standard methods well known to those skilled in the art.

[0120] Example 1: Preparation and characterization of AIE material AF

[0121] The preparation method of AIE material AF is as follows: (4-(naphthyl-1-yl(phenyl)amino)phenyl)boronic acid (CAS: 717888-41-0, 847 mg, 2.5 mmol, 2.0 equivalent) was fully dissolved in 10 mL of ethanol in a reaction system. 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 equivalent) and 2 mL of sodium carbonate aqueous solution (2 M) were added. The mixture was deoxygenated by three consecutive vacuum-nitrogen cycles (each evacuation for 10 minutes followed by nitrogen purging). Subsequently, 5 mL of toluene solution containing tetra(triphenylphosphine)palladium(0) (142 mg, 0.125 mmol, 0.1 equivalent) was injected into the reaction system, and three additional vacuum-nitrogen cycles were performed under the same conditions. The reaction system was vigorously stirred at 85°C for 24 hours under nitrogen protection. 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 carried out by silica gel column chromatography (eluent: n-hexane / dichloromethane = 2 / 1, v / v), finally yielding AIE material AF as a black solid (yield: 33.1%).

[0122] AIE material AF was sampled and dissolved in a deuterated reagent, and then analyzed using a Bruker Avance III 500M superconducting nuclear magnetic resonance spectrometer. 1 H-H-H spectrum and 13 Characterization by C-carbon spectra: 1 H 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- dδ 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. AIE material AF was sampled and dissolved in mass spectrometry-grade acetonitrile. Mass spectrometry characterization was performed using a Waters Xevo G2-XS Qtof time-of-flight tandem high-resolution mass spectrometer: ESI-MS calcd for C 58 H 38 N4S3, [M + H] + 887.2337, found 887.2398.

[0123] Example 2: Characterization of fluorescence properties of AIE material AF

[0124] The optical properties of the AIE material AF were systematically characterized using a UV-Vis spectrophotometer and a fluorescence spectrophotometer. For example... Figure 5 As shown, the AIE material AF dissolved in tetrahydrofuran (THF) exhibits significant absorption and fluorescence emission peaks at 530 nm and 715 nm, respectively, demonstrating 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 effectively reduces self-quenching and lowers the background of biological autofluorescence, thus benefiting bioimaging applications.

[0125] like Figure 6 As shown, the AIE material AF exhibits weak fluorescence emission in pure acetonitrile (100%), with a slight decrease in fluorescence intensity as the water content increases from 0% to 20%. This initial decrease in fluorescence can be attributed to the enhanced solvent polarity promoting the twisted intramolecular charge transfer (TICT) effect. However, when the water content exceeds 20%, the fluorescence intensity significantly increases, confirming that material AF possesses a significant focusing-induced emission (AIE) effect in the aggregated state.

[0126] Structural analysis of AIE material AF, such as Figure 7As shown, the thiophene-benzothiadiazole unit forms large dihedral angles of approximately 73.25°, 63.37°, and 83.82° with the adjacent aromatic benzene ring, respectively. This distorted molecular conformation effectively suppresses π-π stacking interactions in the aggregated state. The aromatic amine group acts as a molecular rotor, restricting intramolecular motion while preserving intramolecular charge transfer (ICT) effects during aggregation. This dual-action mechanism suppresses non-radiative decay pathways while maintaining radiative transitions, ultimately achieving enhanced fluorescence. In summary, the AIE material AF, with its near-infrared emission, large Stokes shift, and AIE properties, shows great potential for developing long-wavelength emission fluorescent nanoparticles.

[0127] Example 3: Preparation and Characterization of Nanoparticles

[0128] The specific preparation methods for AF@F127 NPs, AF@SiO2 NPs, and AF@SiO2-Dex NPs are as follows:

[0129] 1) Add ultra-dry tetrahydrofuran (THF, 1 mL), THF solution containing AIE material AF (50 μL, 1 μM), THF solution containing poloxamer F127 (1 mL, 50 mg / mL), and methyl orthosilicate (TMOS, 0-100 μL) to a 5 mL centrifuge tube, and then homogenize using the ultrasonic probe of a cell disruptor (50 W power, 10 minutes, 10-second on / 10-second off interval).

[0130] When 0 μL of TMOS was added, micellar nanoparticles AF@F127 NPs were prepared for control studies. Subsequent studies found that when the volume of added TMOS was 35 μL, i.e., when TMOS accounted for 1.75% in the above 2 mL THF system, the performance of the obtained nanoparticles AF@SiO2NPs was optimal. The nanoparticles AF@SiO2NPs prepared by this method were used in subsequent tests and applications.

[0131] 2) Add 10 mL of deionized water to a 50 mL round-bottom flask. While stirring at 1500 rpm, add the above tetrahydrofuran mixture dropwise in 50 μL increments. Transfer the resulting water / THF mixture to a 5 mL centrifuge tube and homogenize it again using the sonication probe of a cell disruptor (power: 50W, duration: 10 minutes, 10-second on / 10-second off interval). Transfer the solution back to the round-bottom flask, cover the mouth of the flask with aluminum foil, poke several small holes with a toothpick, and stir at 1500 rpm for 5 days.

[0132] 3) Transfer the solution to a dialysis bag with a molecular weight cutoff of 40 K and dialyze for 3 days to remove residual THF or other water-soluble small molecule impurities. Then, use aqueous filters with pore sizes of 0.45 μm and 0.22 μm to remove large particulate impurities, obtaining a purified aqueous solution of two-photon AIE nanoparticles AF@SiO2NPs.

[0133] The preparation method of AF@SiO2-Dex NPs nanoparticles also includes step 4) after step 3).

[0134] 4) 10 mL of 1M hydrochloric acid and 150 μL of aminopropyltriethoxysilane (APTES) were added to an aqueous solution of AF@SiO2NPs with stirring. After stirring for 24 hours, the mixture was centrifuged at 5000 rpm for 20 minutes using an ultrafiltration centrifuge tube (MWCO 100 kD). The nanoparticles were 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 (molecular weight 1.5K, concentration 5 mM). The mixture was stirred in an ice bath for 12 hours. Then, the mixture was 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.

[0135] NMR characterization of nanoparticles: The samples were lyophilized to remove moisture, resuspended with D₂O as a deuterated reagent, and then subjected to 1H NMR spectroscopy. Figure 8As shown, the NMR signals of AF@F127 NPs mainly originate from poloxamer 407, whose block structure is PEO-PPO-PEO. The characteristic peak at 3.72 ppm is from PEO, while the peaks at 3.58 ppm and 1.19 ppm are from PPO. The 1H NMR signals of AF@SiO2 NPs are similar to those of AF@F127 NPs, with characteristic peaks from PEO (3.72 ppm) and PPO (3.58 ppm and 1.19 ppm). The N-hydroxysuccinimide activated ester (NHS) modified dextran (Dextran-NHS) signals at 5.00, 4.00, 3.93, 3.78, 3.74, 3.60, and 3.54 ppm originate from glucose monomers, while the characteristic peak at 2.74 ppm originates from the NHS group. 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 in the 1H NMR spectrum of AF@SiO2-Dex NPs, and the disappearance of the original characteristic peak belonging to the NHS group (2.74 ppm) suggests that dextran was successfully modified on the silica layer surface of the nanoparticles through amidation coupling.

[0136] Example 4: Effect of silica layer encapsulation on nanoparticle properties

[0137] The physical properties of micellar AIE nanoparticles AF@F127 NPs without a silica layer and nanoparticles AF@SiO2 NPs with a silica layer were compared. Figure 9 As shown, AF@F127 NPs and AF@SiO2 NPs have similar absorption peak wavelengths (520 nm and 523 nm). Figure 10 As shown, the emission peak wavelengths of the two are similar (710 nm and 703 nm). It is noteworthy that, under the same AF concentration, the fluorescence emission intensity of AF@SiO2NPs is significantly enhanced by 3.34 times compared to AF@F127 NPs. Figure 11 As shown, dynamic light scattering (DLS) measurements revealed that AF@F127 NPs and AF@SiO2 NPs had similar hydrodynamic particle sizes, 25.91 ± 3.77 nm and 28.87 ± 2.14 nm, respectively, indicating 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 exhibited an amorphous morphology. Figure 12 ); while AF@F127 NPs exhibit a distinct concentric circle morphology, with a silicon shell thickness of approximately 8.31 ± 0.74 nm ( Figure 13 ).like Figure 14As shown, the zeta potential of AF@F127 NPs is relatively small, at -0.54±0.09 mV, due to the electroneutrality of Pluronic F127; while the zeta potential of AF@SiO2 NPs is increased, at -24.22±0.51 mV, due to the protonation of silanol groups on the surface of the silicon dioxide layer.

[0138] Example 5: Optimization of silica encapsulation to enhance the fluorescence properties of AIE nanoparticles

[0139] In the preparation of nanoparticles, different amounts (0-100 μL) of TMOS were introduced using a controlled variable method to optimize the fluorescence performance of the nanoparticles. For example... Figure 15 As shown, when the TMOS addition amount was 0-35 μL, the AF@SiO2NPs solution remained clear, and DLS measurements showed that the hydrodynamic particle size of the nanoparticles was approximately 25 nm, indicating that the encapsulation of the silica shell around the hydrophobic core of the AIE did not significantly alter its hydrodynamic properties. When the TMOS addition amount 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 with the hydrolyzed TMOS, leading to aggregation and eventual precipitation. Figure 16 TEM images show that as the TMOS concentration increases, the silicon dioxide layer thickens and exhibits significant particle aggregation. Figure 17 When the amount of TMOS added reaches 70-100 μL, a pink precipitate appears in the solution due to the hydrolysis of excess TMOS, and the supernatant becomes colorless, indicating that there is no AIE nanoparticle dispersion in the supernatant. Figure 18 This study investigated the effect of silica encapsulation on the fluorescence properties of AF@SiO2NPs, with the control group consisting of micellar nanoparticles AF@F127 NPs without silica encapsulation (i.e., with 0 μL of TMOS added). Within the range of 0–35 μL (0–1.75%) of TMOS added, the fluorescence enhancement factor of the AF@SiO2NPs nanoparticles (reaching a maximum of 3.13 times at 35 μL of TMOS) exhibited a dose-dependent effect. This is attributed to the centripetal compression effect of the silica layer on the hydrophobic AIE core, effectively enhancing the AIE effect. When the TMOS addition increased to 40–100 μL, despite significant particle aggregation and precipitation, the overall fluorescence intensity of the solution remained relatively stable, suggesting that once the outwardly thickened silica shell reached a certain thickness, it could not exert further centripetal pressure 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 good hydrodynamic and fluorescence properties.

[0140] Example 6: Effect of silica encapsulation on the two-photon properties of nanoparticles

[0141] The above-mentioned optimization strategy (adding 35 μL of TMOS, accounting for 1.75% of the system) was used to prepare AF@SiO2NPs, and their two-photon performance was compared with that of AF@F127NPs without silica encapsulation. Figure 19 As shown, the absolute fluorescence quantum yield (QY = the ratio of emitted photons to absorbed photons) was determined using 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, with the latter being 3.99 times that of the former, indicating that silica encapsulation effectively enhances the fluorescence performance of the nanoparticles.

[0142] To investigate the effect of silica encapsulation on the nonlinear optical properties of AIE nanoparticles, such as... Figure 20 As shown, methanol samples containing AF@F127 NPs or AF@SiO2 NPs in a capillary sample tube were irradiated using a Leica SP8 DIVE multiphoton confocal microscope equipped with a Coherent Chameleon two-photon laser system, with Rhodamine B (RB) as a reference. Fluorescence images of the samples in the glass capillary tube were acquired and fluorescence intensity values ​​were calculated under femtosecond laser excitation in the 820-1080 nm wavelength range. The fluorescence intensity of the nanoparticles AF@F127 NPs and AF@SiO2 NPs showed a linear relationship with the logarithm of the excitation power, with R² values ​​of 0.9958 and 0.9954, respectively, suggesting two-photon optical properties. Figure 21 ).like Figure 22 As shown, in the near-infrared spectral range of 820-1080 nm, AF@SiO2NPs exhibit a significantly enhanced two-photon absorption cross section (2PACS), especially showing a characteristic peak at 1060 nm with an absorption cross section of 256.56±14.72 GM, which is 4.37 times stronger than that of AF@F127 NPs (49.63±8.48 GM). This suggests that AF@SiO2NPs can achieve more efficient excitation with significantly reduced laser power. The reduced excitation power requirement not only helps to reduce photobleaching of nanoparticles but also effectively reduces phototoxic damage to biological tissues, thereby greatly improving the biocompatibility of the system in long-term imaging applications.

[0143] Example 7: Performance characterization of dextran-modified nanoparticles AF@SiO2-Dex NPs

[0144] Furthermore, dextran (1.5K) was modified onto the silica layer surface of AF@SiO2NPs to enhance the biocompatibility of the nanoparticles. For example... Figure 23 As shown, TEM results indicate that AF@SiO2-Dex NPs possess a core-shell structure similar to AF@SiO2 NPs. Figure 24 As shown, 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 is comparable to that of AF@SiO2 NPs, indicating that dextran modification did not affect the fluorescence performance of the nanoparticles. Figure 25 As shown, the hydrodynamic particle size of AF@SiO2-DexNPs measured by DLS was 33.96 ± 3.03 nm. Figure 26 As shown, the zeta potential of AF@SiO2-Dex NPs was measured to be -0.73±0.12 mV. Compared with AF@SiO2NPs (-24.22±0.51 mV), the absolute value of the zeta potential was significantly reduced, indicating that it can better avoid protein adsorption and further improve biocompatibility.

[0145] Example 8: Comparison of the stability of nanoparticles

[0146] like Figure 27 As shown, equal volumes (10 μM) of aqueous solutions of nanoparticles AF@F127 NPs, AF@SiO2 NPs, and AF@SiO2-Dex NPs were allowed to stand for one month. The supernatant was collected every 5 days, and the fluorescence intensity was detected using a fluorescence spectrophotometer. It was observed that the fluorescence of the supernatant in the AF@F127 NPs group gradually decreased compared to day 0, and a small purple precipitate gradually appeared in the solution. This is presumably due to the breakdown of the micellar nanoparticles AF@F127 NPs, precipitating out the AIE material AF, which ultimately led to the gradual decrease in the fluorescence intensity of the supernatant. The fluorescence intensity of the AF@SiO2 NPs and AF@SiO2-Dex NPs groups remained essentially unchanged, demonstrating better stability.

[0147] Example 9: Cytotoxicity of AF@SiO2-Dex NPs

[0148] To thoroughly evaluate the biocompatibility of AF@SiO2-Dex NPs, a nanoparticle combining the aforementioned materials, experiments were conducted using classic immortalized HeLa cells and HUVEC cells, which are crucial for angiogenesis. Figure 28As shown, the two cell types were treated with different concentrations (0-50 μM) of AF@SiO2-Dex NP nanoparticles for 48 hours, and cell viability was detected using a CCK8 assay kit. The results showed that both cell types maintained extremely high viability (>95%) after treatment with AF@SiO2-Dex NP nanoparticles, indicating that AF@SiO2-Dex NP nanoparticles have minimal cytotoxicity and high biocompatibility.

[0149] Example 10: In vivo safety assessment of AF@SiO2-Dex NPs nanoparticles

[0150] To assess the in vivo safety of AF@SiO2-Dex NP nanoparticles, female C57BL / 6 mice (6-7 weeks old) were intravenously injected with AF@SiO2-Dex NP nanoparticles (100 μL, dose groups 0, 5, and 50 μM) on days 1 and 7 of a 14-day experimental cycle. Figure 29 As shown, 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 feeding behavior, locomotor activity, or excretory function of the experimental animals. Mice were sacrificed on day 14, and major organs were collected and weighed. The organ coefficient (organ / body weight ratio) was calculated. The results showed no significant difference between the experimental groups and the control group. Figure 30 ), Figure 31 Key hematological parameters were measured in blood samples collected from mice on day 14. Results showed that white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red blood cell distribution width coefficient of variation (RDW-CV) were all within physiological ranges, confirming normal hematopoietic function and the absence of hepatotoxicity or nephrotoxicity. Pathological evaluation of fixed sections from vital organs (heart, liver, spleen, lung, kidney, and uterus) showed normal tissue structure, with no inflammatory infiltration or pathological changes. Figure 32 The above comprehensive evidence confirms that AF@SiO2-Dex NPs nanoparticles have good biocompatibility and potential for biomedical applications.

[0151] Example 11: Visualization of uterine blood vessels in mice

[0152] AF@SiO2-Dex NP nanoparticles possess high biocompatibility, high fluorescence quantum efficiency, and high two-photon absorption cross-section, making them highly promising for bioimaging applications. Here, to investigate the dynamics of blood vessels in the early pregnancy or non-pregnant uterus, AF@SiO2-Dex NP nanoparticles were used to perform fluorescence visualization imaging of the uterine vascular network in mice.

[0153] 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 exposed. Following intravenous injection of AF@SiO2-Dex NPs into the mouse tail vein, imaging was performed using a 25x objective lens of a two-photon microscope. Based on the collagen-rich characteristics of the uterine serosa and myometrium, the uterine tissue was excited at 960 nm using a two-photon microscope, and the typical bundled structure of collagen fibers was clearly displayed in second harmonic generation (SHG) imaging mode. Figure 33 (a) The 960 nm excitation light mentioned above can effectively excite the near-infrared fluorescence of AF@SiO2-DexNP nanoparticles in the first region, enabling visualization of vascular networks. Figure 33 (b) in the middle.

[0154] Notably, the SHG signal intensity within the vascular lumen is significantly attenuated compared to the surrounding matrix tissue. This phenomenon stems from the inherent optical properties of blood: as a homogeneous liquid phase primarily composed of water and cells, blood lacks the collagen matrix structure required for efficient SHG emission. Therefore, the vascular contour precisely occupies the dendritic low-signal region in the SHG image (…). Figure 33 (c) The above spatial complementarity results validate the ability of SHG imaging combined with two-photon imaging of nanoparticles AF@SiO2-Dex NPs to resolve the spatial relationship between the vascular system and the extravascular matrix.

[0155] As imaging depth increases, such as Figure 33 As shown in d, SHG imaging of the endometrial layer reveals multiple cavity-like structures, which are endometrial glands. Figure 33 e and Figure 33 The f-image shows that AF@SiO2-Dex NPs can image the capillary network (4-10 μm in diameter) around the glands. This was achieved by analyzing SHG (… Figure 33 g) and AF@SiO2-Dex NPs ( Figure 33 The z-axis layer was scanned using the h) channel, enabling three-dimensional reconstruction of uterine tissue at a depth of over 300 μm. Figure 33 (i) Nanoparticles AF@SiO2-DexNPs exhibit excellent optical penetration properties, enabling two-photon depth imaging and providing a comprehensive research tool for resolving the spatial relationships of heterogeneous uterine structures.

[0156] Example 12: AF@SiO2-Dex NPs used to study vascular permeability

[0157] Fluorescently labeled dextran (such as FITC-glucan) is widely used in vascular permeability studies due to its good biodegradability and low immunogenicity. Here, mice were injected via tail vein into nanoparticles AF@SiO2-Dex NPs with FITC-glucan, followed by two-photon imaging of the mouse uterus. In the early stages of injection, collagen fibers were visualized against a background of SHG imaging. Figure 34 In part a), the nanoparticles AF@SiO2-Dex NPs and FITC-glucan are both locally distributed within the blood vessel interior. 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 they had overlapping distribution patterns. Figure 35 ).

[0158] Figure 36 The image shows collagen fibers against a background as visualized by SHG imaging approximately 60 minutes after injection. Figure 36 (a) The AF@SiO2-Dex NPs nanoparticles still maintain their sustained vascular retention properties and stable fluorescence intensity, indicating their potential as a real-time fluorescent angiography agent. Figure 36 (b) In contrast, FITC-glucan shows a gradual decrease in signal intensity within blood vessels, accompanied by significant extravasation into perivascular tissues. Figure 36 (cg in the middle). At this time, the colocalization coefficient decreased significantly 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-glucan to study the mechanisms of uterine vascular transport and diffusion.

[0159] 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 within the protection scope of the present invention.

Claims

1. A method for preparing two-photon AIE nanoparticles, characterized in that, Includes the following steps: S1. Tetrahydrofuran, a tetrahydrofuran solution containing AIE material, a tetrahydrofuran solution containing poloxamer 407, and methyl orthosilicate are mixed to obtain a mixture, wherein the AIE material is AF, and the structure of the AF is as follows: The volume ratio of the tetrahydrofuran, the tetrahydrofuran solution containing 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 AIE material is 0.9-1.1 μM, and the concentration of the tetrahydrofuran solution containing poloxamer 407 is 45-55 mg / mL. S2. Add the mixture obtained in S1 dropwise to the deionized water while stirring; S3. Dialysis and filtration were performed to obtain purified two-photon AIE nanoparticles AF@SiO2 NPs.

2. The preparation method according to claim 1, characterized in that, Following step S3, step S4 is further included: the two-photon AIE nanoparticles AF@SiO2 NPs are added to hydrochloric acid and aminopropyltriethoxysilane under stirring, stirred and then subjected to two ultrafiltrations, resuspended in PBS, added to PBS containing N-hydroxysuccinimide activated ester modified dextran, stirred in an ice bath, ultrafiltered, resuspended in ultrapure water, and ultrafiltered again to obtain purified two-photon AIE nanoparticles AF@SiO2-DexNPs.

3. The preparation method according to claim 2, characterized in that, In step S4, the volume ratio of the two-photon AIE nanoparticles AF@SiO2 NPs, 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 0.9-1.1M, and the concentration of the dextran is 4.5-5.5mM. And / or, the specific operation of the ultrafiltration is as follows: using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kD, centrifuging at 5000 rpm for 18-22 minutes; Add hydrochloric acid and aminopropyltriethoxysilane, stir for 22-26 h, or stir in an ice-water bath for 10.8-13.2 h.

4. The preparation method according to claim 1, characterized in that, The preparation method of the AF includes the following steps: (1) Dissolve (4-(naphthyl-1-yl(phenyl)amino)phenyl)boronic acid completely in ethanol, add toluene solution containing 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole and sodium carbonate aqueous solution, and then perform deoxygenation treatment; (2) Inject a toluene solution containing tetra(triphenylphosphine)palladium(0) and perform deoxygenation treatment; (3) Stir vigorously at 85-95℃ for more than 24 hours under nitrogen protection, cool to room temperature, and then rotary evaporate to obtain crude product; (4) The crude product was dissolved in dichloromethane and recrystallized in n-hexane, and then purified by silica gel column chromatography to obtain the final product AF.

5. The preparation method according to claim 4, characterized in that, The equivalent ratio of (4-(naphthyl-1-yl(phenyl)amino)phenyl)boronic acid, 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole and tetra(triphenylphosphine)palladium(0) is (18-22):(9-11):1; The molar volume ratio of (4-(naphth-1-yl(phenyl)amino)phenyl)boronic acid to ethanol is 0.25 mmol / mL, the molar volume ratio of 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).

6. The preparation method according to claim 4, characterized in that, The specific operation of the deoxygenation process is as follows: vacuuming, filling with nitrogen after 10 minutes, and repeating this cycle 3 times. And / or, the eluent used in the silica gel column chromatography purification is n-hexane and dichloromethane in a volume ratio of 2:

1.

7. The preparation method according to claim 1, characterized in that, After mixing in step S1 and adding to the deionized water in step S2, a homogenization process is also included. The specific operation of the homogenization process is as follows: the ultrasonic probe of the cell disruptor is used for homogenization, with a power of 50W and a duration of 10 minutes, with an interval of 10 seconds on and 10 seconds off. And / or, in step S2, the stirring speed of the deionized water is 1500 rpm, and the stirring speed after uniform treatment in step S2 is 1500 rpm for 5 days. And / or, before stirring after uniform treatment in step S2, the solution is further transferred to a container, and aluminum foil is placed over the bottle opening with multiple small holes punched through it. And / or, the specific operation of dialysis in step S3 is as follows: transfer the solution obtained in S2 into a dialysis bag with a molecular weight cutoff of 40K, and dialyze for 3 days; And / or, the specific operation of filtration in step S3 is as follows: use a water-based syringe filter with a pore size of 0.45μm for filtration, and then use a water-based syringe filter with a pore size of 0.22μm for filtration.

8. A two-photon AIE nanoparticle, characterized in that, It is prepared by the preparation method described in any one of claims 1-7.

9. The application of the two-photon AIE nanoparticles as described in claim 8 in the preparation of contrast agents for deep imaging of biological tissues.

Images