Preparation method and application of aie probe for long-term two-photon imaging of cerebral vascular system of conscious mice

Abstract

This invention discloses a method for preparing and applying an AIE probe for long-term two-photon imaging of the cerebrovascular system in awake mice. The probe is prepared by assembling an AIE precursor with DSPE-mPEG2000-Mal into nanoparticles, followed by coupling with the cell-penetrating peptide TAT. This probe exhibits excellent photostability, long-term vascular retention, good biocompatibility, and neuroimmunocompatibility. In awake mouse models, it can achieve three-dimensional imaging of cerebrovascular systems at depths exceeding 500 micrometers and stably labels blood vessels for up to 35 days without activating microglia. This probe supports multi-scale imaging from acute hemodynamics to long-term structural remodeling, providing a powerful tool for neurovascular research.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials and neuroimaging technology, specifically relating to a method for preparing and applying a focusing-induced emission (AIE) probe for long-term two-photon imaging of the cerebrovascular system in awake mice. Background Technology

[0002] The dynamic changes in the structure and function of the cerebral vascular network are key to understanding neurovascular coupling, vascular pathology, and the mechanisms of cerebral blood flow regulation. Two-photon microscopy, with its deep tissue penetration and high spatiotemporal resolution, has become an important tool for studying living cerebral blood vessels. However, achieving long-term, stable, and high-resolution imaging of cerebral blood vessels still faces two major challenges: First, fluorescent probes have short vascular retention times, and traditional small-molecule dyes (such as fluorescein and rhodamine) are easily and rapidly cleared from blood vessels, exhibiting poor photostability and failing to meet the requirements for long-term imaging. Second, although nanoprobes can prolong circulation time, they often leak outside the blood vessels due to insufficient endothelial affinity, and some nanomaterials are unstable under physiological conditions, potentially triggering immune responses.

[0003] Aggregation-induced emitters (AIEgens) have attracted widespread attention in the field of bioimaging in recent years due to their advantages such as enhanced emission in the aggregated state, strong resistance to photobleaching, and good biocompatibility. However, most AIE probes still require optimization of their surface properties to improve their retention capacity in blood vessels and the stability of long-term imaging. In particular, for long-term imaging in awake mice, the probes need to withstand repeated high-power laser excitations without interfering with the immune homeostasis of the central nervous system.

[0004] Therefore, developing an AIE probe that integrates high photostability, excellent vascular retention, good biocompatibility, and neuroimmunocompatibility is of great significance for advancing basic research on cerebrovascular diseases and longitudinal studies of disease models. Summary of the Invention

[0005] The present invention aims to provide an AIE probe suitable for long-term two-photon imaging of the cerebrovascular system in awake mice and its preparation method, so as to solve the problems of short vascular retention time, poor photostability, poor biocompatibility and neuroimmunological compatibility of existing fluorescent probes.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0007] A method for preparing an AIE probe for long-term two-photon imaging of the cerebrovascular system in awake mice includes the following steps:

[0008] (1) Maleic anhydride-triphenylamine and N-(4-aminobutyl)-N-ethyl isoluminol were co-dissolved in the organic solvent N,N-dimethylformamide, and the reaction was carried out in an air atmosphere at 150-170 °C for 6-10 hours using triethylamine catalyst. The resulting reaction solution was first extracted with dichloromethane and then dried with anhydrous magnesium sulfate. The crude product was purified by silica gel column chromatography to obtain the AIE precursor compound.

[0009] (2) The AIE precursor compound and DSPE-mPEG2000-Mal were co-dissolved in the organic solvent tetrahydrofuran. The resulting mixture was injected into water for ultrasonic emulsification. Then the tetrahydrofuran was removed to obtain AIE nanoparticles with maleimide groups on the surface.

[0010] (3) The AIE nanoparticles were reacted with the cell-penetrating peptide TAT, coupled by thioether bond, and then purified to obtain TAT NPs.

[0011] In step (2) of the preparation method, the mass ratio of the AIE precursor compound to DSPE-mPEG2000-Mal is 1:2.

[0012] The amino acid sequence of the cell-penetrating peptide TAT in step (3) of the preparation method is RKKRRQRRRC.

[0013] The purification in step (3) of the preparation method is carried out by ultrafiltration, and the molecular weight cutoff value of the ultrafiltration membrane is 100 kDa.

[0014] An AIE probe obtained by the preparation method described above.

[0015] The application of the aforementioned AIE probe in the preparation of a reagent for long-term two-photon imaging of the cerebrovascular system in awake mice.

[0016] A kit for long-term two-photon imaging of the cerebrovascular system in awake mice, comprising the AIE probe described above.

[0017] The beneficial effects of this invention are:

[0018] The AIE probe for long-term two-photon imaging of the cerebrovascular system in awake mice of the present invention has the following advantages compared with the prior art:

[0019] (1) Excellent colloidal stability and biocompatibility: TAT-NPs maintain stable particle size and polydispersity index under different pH conditions, hemolysis rate is less than 2%, no obvious cytotoxicity to various cell lines, no pathological damage to major organs of mice, and normal serum biochemical indicators, indicating that it has good blood compatibility and in vivo safety.

[0020] (2) Excellent photostability: TAT-NPs exhibit extremely high resistance to photobleaching under both low-frequency (1 / 180 Hz) and high-frequency (2.27 Hz) two-photon excitation. After continuous imaging for 60 minutes or 660 seconds, they still maintain more than 90% of the initial fluorescence intensity, which is far superior to traditional dextran probes.

[0021] (3) Long-term vascular retention capacity: The introduction of TAT peptides significantly enhances the affinity between nanoparticles and vascular endothelial cells, greatly prolonging the retention time of the probe in blood vessels. 120 minutes after injection, the fluorescence intensity of TAT-NPs in arterioles and capillaries remained above 90% and 95%, respectively, while commercial probes had decayed to below 50%; vascular structures could still be clearly distinguished 96 hours after injection, meeting the needs of long-term tracking.

[0022] (4) High neuroimmune compatibility: In the Cx3cr1-GFP transgenic mouse model, TAT-NPs can achieve vascular labeling for up to 35 days, and microglia always maintain a resting branched morphology. Cell density, cell body area and Cd68 expression level have not changed significantly, proving that the probe does not disrupt the immune homeostasis of the central nervous system.

[0023] (5) Supports multi-scale dynamic monitoring: TAT-NPs are not only suitable for acute hemodynamic imaging (such as real-time changes in vessel diameter), but also for tracking long-term vascular structural remodeling, combining the advantages of functional and structural imaging.

[0024] (6) Simple and efficient preparation method: The preparation method of this invention is simple to operate and the reaction conditions are mild. First, the synthesis of the AIE precursor is carried out in an air atmosphere, without the need for inert gas protection, which reduces the requirements for reaction conditions; second, the assembly of nanoparticles adopts a one-step injection sonication method, avoiding complex microfluidic or membrane hydration steps; finally, unreacted TAT peptides and organic solvents can be efficiently removed by ultrafiltration purification, without the need for cumbersome operations such as column chromatography. The entire preparation process is low in cost, easy to scale up for production, and has good industrialization prospects. Attached Figure Description

[0025] Figure 1 Mass spectrum of the AIE molecule in Example 1.

[0026] Figure 2 Dynamic light scattering (DLS) analysis diagram of TAT-NPs.

[0027] Figure 3 Transmission electron microscopy (TEM) image showing the spherical morphology of TAT-NPs.

[0028] Figure 4 Zeta potential measurement results for NPs and TAT-NPs.

[0029] Figure 5 The changes in particle size (top) and PDI (bottom) of TAT-NPs under different pH conditions.

[0030] Figure 6 The UV-Vis absorption spectrum (upper curve) and photoluminescence emission spectrum (lower curve) of TAT-NPs.

[0031] Figure 7 CCK-8 assay was used to evaluate the cell viability of TAT-NPs at different concentrations.

[0032] Figure 8 Results of hemolysis experiments of TAT-NPs at different concentrations (25-400 μg / mL).

[0033] Figure 9 Serum biochemical parameters of mice 7 days after a single dose of TAT-NPs.

[0034] Figure 10 H&E staining sections of major organs 7 days after a single retroorbital injection of TAT-NPs or PBS.

[0035] Figure 11 Z-Stack imaging results (a) and image post-processing and analysis workflow (b).

[0036] Figure 12 In vivo two-photon fluorescence images of the superficial cortex of mice after injection of TAT-NPs, FITC-glucan, and TRITC-glucan.

[0037] Figure 13 and Figure 12 Corresponding in vivo fluorescence images of the deep cortex under the same probe and time conditions.

[0038] Figure 14 Quantitative analysis of the relative fluorescence intensity of TAT-NPs, FITC-glucan, and TRITC-glucan in cortical arterioles within 120 minutes after injection in mice.

[0039] Figure 15 Quantitative analysis of the relative fluorescence intensity of three probes in cortical capillaries within 120 minutes after injection in mice.

[0040] Figure 16 In vivo fluorescence images of mouse arterioles (left panel) and capillaries (right panel).

[0041] Figure 17 Quantitative analysis of the relative fluorescence intensity of three probes in arterioles (left) and capillaries (right) in mice 0-4 hours after injection.

[0042] Figure 18 and Figure 16 Combined images of TAT-NPs (red channel) and FITC-glucan (green channel) in mouse arterioles and capillaries at the corresponding imaging time points.

[0043] Figure 19 Quantitative analysis of the relative fluorescence intensity of TAT-NPs and FITC-glucan in arterioles (left) and capillaries (right) after injection in mice.

[0044] Figure 20 Time-series images of live two-photon imaging of TAT-NPs photobleached at a scan frequency of 1 / 180 Hz.

[0045] Figure 21 The relative fluorescence intensity of TAT-NPs during the period of 0-60 minutes was scanned at a frequency of 1 / 180 Hz (corresponding to...). Figure 20 Quantitative analysis of ).

[0046] Figure 22 TAT-NPs in vivo two-photon imaging of mouse photobleaching at a scan frequency of 2.27 Hz.

[0047] Figure 23 The relative fluorescence intensity of TAT-NPs during the period of 0-660 seconds was scanned at a frequency of 2.27 Hz (corresponding to...). Figure 22 The dynamic changes of ).

[0048] Figure 24 Short-term (0-8 hours) and long-term (0-96 hours) in vivo two-photon imaging time series of TAT-NPs photobleaching.

[0049] Figure 25 Supplemental short-term (0-8 hours) and long-term (0-96 hours) in vivo two-photon imaging sequences for TAT-NPs photobleaching.

[0050] Figure 26 correspond Figure 24 Spatial fluorescence intensity distribution.

[0051] Figure 27 correspond Figure 25 Quantitative analysis of relative fluorescence intensity.

[0052] Figure 28 Longitudinal dual-channel in vivo imaging of a mouse (a) and a magnified view of the green channel (b).

[0053] Figure 29 Quantitative curves of relative fluorescence intensity of TAT-NPs in mice within 0-35 days after injection.

[0054] Figure 30 Dynamic changes in the number of microglia in each field of view within 0-35 days after mouse injection.

[0055] Figure 31 Results of immunofluorescence staining in mice.

[0056] Figure 32 Sholl analysis chart.

[0057] Figure 33 Cell body area of ​​microglia.

[0058] Figure 34 The ratio of Cd68 to Iba-1 fluorescence intensity.

[0059] Figure 35 In vivo imaging of sensory cortical vessels labeled with TAT-NPs (a) and Z-score dynamic curves corresponding to four vascular regions (b). Detailed Implementation

[0060] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the parts mentioned in the embodiments refer to parts by mass, and the percentages refer to percentages by mass.

[0061] Example 1: Preparation of AIE probes for long-term two-photon imaging of the cerebrovascular system in awake mice.

[0062] The molecular formula of the AIE probe is C 54 H 46 N6O4 has the following structural formula:

[0063]

[0064] (1) Synthesis of AIE molecules:

[0065] Maleic anhydride-triphenylamine (MAH-TPA, 584.0 mg, 1.0 mmol) and N-(4-aminobutyl)-N-ethylisoluminol (ABEI, 331.4 mg, 1.2 mmol) were dissolved in 20 mL of N,N-dimethylformamide, and 0.1 mL of triethylamine catalyst was added. The mixture was heated to 160 °C in air and reacted for 8 hours.

[0066] After the reaction was completed, the reaction solution was extracted three times with dichloromethane, the organic phases were combined, dried with anhydrous magnesium sulfate, filtered, and the solvent was removed by rotary evaporation.

[0067] The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 20:1, v / v), and the product was a dark red powder.

[0068] High-resolution mass spectrometry (HRMS) analysis of the product revealed a peak at m / z 842.35979, which is similar to that of compound C. 54 H 46 The exact molecular weight of N6O4 (theoretical value 842.35806) matches highly in the figure. Additionally, the figure also shows a peak at m / z 843.36714, corresponding to its [M+H] content. + The presence of the ion (theoretical value 843.36589) further supports the conclusion that this product is the target compound. The mass spectrum of the product is shown below. Figure 1 .

[0069] (2) Preparation of nanoparticles

[0070] The product obtained in step (1) (1 mg) and DSPE-mPEG2000-Mal (2 mg) were dissolved together in 1 mL of tetrahydrofuran (THF) to obtain a clear solution. Under vigorous stirring, the solution was rapidly injected into 10 mL of pure water using a syringe, followed by strong sonication for 60 seconds (100 W) to ensure uniform dispersion. The resulting suspension was stirred at room temperature in a fume hood for 12 hours to allow complete evaporation of THF.

[0071] Subsequently, 2 mg of TAT peptide (sequence: RKKRRQRRRC, purity >95%) was added, and stirring was continued for 12 hours to allow the peptide to form a stable thioether bond with the maleimide group on the surface of the nanoparticles through the thiol group of its C-terminal cysteine ​​residue. After the reaction was completed, the nanoparticle suspension was transferred to an ultrafiltration tube (molecular weight cutoff 100 kDa), centrifuged at 6000 rpm for 1 hour, and repeated three times to remove unreacted TAT peptide and organic solvent. Finally, the suspension was resuspended in PBS to obtain purified AIE-2p-920@TAT NPs (hereinafter referred to as TAT-NPs), which were stored at 4 °C for later use.

[0072] Example 2: Characterization of the physicochemical properties of TAT-NPs

[0073] 1. Particle size and zeta potential determination

[0074] An appropriate amount of TAT-NPs suspension was taken and diluted with deionized water to a suitable concentration. The hydrated particle size, polydispersity index (PDI), and zeta potential were measured using a dynamic light scattering particle size analyzer (Malvern Zetasizer Nano ZS). The results showed that the average particle size of TAT-NPs was 56.7 nm, and the PDI was 0.269 (…). Figure 2 This indicates a uniform particle size distribution. Zeta potential measurements showed that the unmodified AIE nanoparticles were negatively charged (-21.5 mV), while the TAT-NPs were positively charged (+7.07 mV). Figure 4 This confirms that TAT was successfully coupled to the particle surface.

[0075] 2. Observation using transmission electron microscopy

[0076] The TAT-NPs suspension was dropwise added onto a copper mesh support film and allowed to dry naturally. The morphology was then observed using a transmission electron microscope (TEM, JEOL JEM-1400). The images showed that the TAT-NPs were uniformly spherical, well-dispersed, and the particle size was consistent with the DLS results. Figure 3 ).

[0077] 3. pH stability assessment

[0078] TAT-NPs were placed in buffer solutions with pH values ​​of 4.0, 5.0, 6.0, 7.4, 8.0, 9.0, and 10.0, respectively, and incubated at room temperature for 24 hours before measuring particle size and PDI. The results showed that there were no significant changes in particle size and PDI under any pH conditions. Figure 5 This demonstrates that TAT-NPs possess good colloidal stability.

[0079] 4. Measurement of optical properties

[0080] The absorption spectrum of TAT-NPs was measured using a UV-Vis spectrophotometer (Shimadzu UV-2600), showing a broad absorption peak in the 400-600 nm range, attributed to intramolecular charge transfer transitions. The emission spectrum was measured using a fluorescence spectrophotometer (Hitachi F-7000) with an excitation wavelength of 800 nm (two-photon equivalent), showing an emission peak around 660 nm. Figure 6 This indicates that the probe is suitable for near-infrared two-photon imaging.

[0081] Example 3: Biocompatibility evaluation of TAT-NPs

[0082] 1. Cytotoxicity assay (CCK-8 assay)

[0083] Human umbilical vein endothelial cells (HUVECs), human lung epithelial cells (BEAS-2B), human proximal renal tubular cells (HK-2), and human hepatic stellate cells (LX-2) were seeded into 96-well plates and cultured for 24 hours. Different concentrations of TAT-NPs (0, 25, 50, 100, 200, and 400 μg / mL) were then added, and the cells were cultured for another 24 hours. Next, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for 2 hours. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. The results showed that the viability of all cells at a concentration of 400 μg / mL was higher than 90%, with no significant cytotoxicity. Figure 7 ).

[0084] 2. Hemolysis test

[0085] Fresh mouse blood was collected, and erythrocytes were collected by centrifugation. The cells were washed three times with PBS and resuspended. Different concentrations (25, 50, 100, 200, 400 μg / mL) of TAT-NPs were mixed with the erythrocyte suspension and incubated at 37 °C for 1 hour. The supernatant was collected by centrifugation, and its absorbance at 540 nm was measured. Deionized water was used as a positive control (100% hemolysis), and PBS was used as a negative control (0% hemolysis). The results showed that the hemolysis rate of TAT-NPs at all concentrations was less than 2%. Figure 8 It meets the safety standard of less than 5% for hemolysis rate specified in the regulations for biological materials.

[0086] 3. In vivo biosafety evaluation

[0087] Twelve C57BL / 6J mice were randomly divided into two groups of six each, with half males and half females. The experimental group received an injection of 50 μL of TAT-NPs (2 mg / mL) via the retroorbital vein, while the control group received an equal volume of PBS. Seven days later, blood samples were collected for serum biochemical analysis (ALT, AST, ALB, ALP, BUN, CREA, UA). Mice were then sacrificed, and the heart, liver, spleen, lungs, kidneys, and brain were collected for H&E staining. Unpaired two-tailed t-tests were used, and bar charts with independent data points show the mean ± standard error. "ns" indicates no significant difference between groups.

[0088] The results showed no significant differences in serum biochemical parameters between the two groups of mice. Figure 9 The morphology of all organs and tissues was normal, and no obvious pathological changes were observed. Figure 10 This indicates that TAT-NPs have good biocompatibility in vivo.

[0089] Example 4: Cerebral angiography process in awake mice and 3D visualization of TAT-NPs

[0090] 1. Animal models and imaging procedures

[0091] C57BL / 6J mice or Cx3cr1-GFP transgenic mice (8-12 weeks old) were used in the experiment according to the following procedure: Day 0: Craniotomy was performed to implant a glass craniotomy window above the cerebral cortex of the mice. Days 7-15: Mice were trained to adapt to the imaging environment by restoring their heads to a fixation device for 20 minutes daily. Day 16: TAT-NPs (50 μL, 2 mg / mL) were injected via the retroorbital vein and immediately placed under a two-photon microscope for imaging. Imaging intervals ranged from 20 minutes to 5 days, used for acute dynamics and long-term structural monitoring, respectively.

[0092] 2. Three-dimensional imaging and reconstruction

[0093] Two-photon microscopy (Olympus FVMPE-RS) with a 25× water immersion objective and an excitation wavelength of 920 nm was used to acquire Z-stack images (step size 2 μm, depth up to 500 μm or more). Three-dimensional reconstruction and layered projection were performed using ImageJ software. Results showed that TAT-NPs could clearly label the cerebral vascular network, with an imaging depth of up to 516 μm. Figure 11 a) Uniform vascular signals can be observed in the superficial (0-100 μm), intermediate (200-300 μm), and deep (400-500 μm) layers.

[0094] Figure 11 (a) Z-Stack imaging results, imaging depth 516 μm (relative to the pia mater). The figure shows the 3D reconstructed image and layered images (0–100 μm, 200–300 μm, and 400–500 μm subpia mater); scale bar is 50 μm.

[0095] 3. Image Post-processing Workflow

[0096] To facilitate quantitative analysis, a standard image processing workflow was established: original image → contrast enhancement → Gaussian smoothing → thresholding → tubular structure extraction → skeletonization. This workflow can effectively extract vascular network parameters, such as density and number of branches. Figure 11 (b) represents the image processing steps.

[0097] Example 5: Comparison of vascular persistence between TAT-NPs and commercial probes

[0098] 1. Short-term imaging comparison

[0099] Mice were randomly divided into three groups (n=4), injected with TAT-NPs (red channel), FITC-glucan (70 kDa, green channel), and TRITC-glucan (70 kDa, red channel), respectively. Two-photon imaging was initiated immediately after injection, and images of the superficial cortex (0-30 μm) and deep cortex (70-100 μm) were acquired at 0, 20, 40, 60, 80, 100, and 120 minutes. Results showed that at 120 minutes, the blood vessels labeled with TAT-NPs still maintained clear outlines, while the two glucan probes had significantly decayed. Figure 12 , 13 ).

[0100] Figure 12 In vivo two-photon fluorescence images of the superficial cortex (0-30 μm subpia mater) after mice were injected with TAT-NPs (red channel), FITC-glucan (green channel, commercial probe), and TRITC-glucan (red channel, commercial probe).

[0101] Figure 13 In Figure 12 Under the same probe and time conditions, corresponding in vivo fluorescence images of the deep cortex (70–100 μm subpia mater), scale bar = 50 μm.

[0102] Quantitative analysis showed that in arterioles, the fluorescence intensity of TAT-NPs remained above 90% after 120 minutes, while FITC-glucan decreased to approximately 50% and TRITC-glucan decreased to approximately 40%. Figure 14 In capillaries, TAT-NPs retained approximately 95%, while the two commercial probes decreased to approximately 60% and 50%, respectively. Figure 15 Statistical analysis showed significant differences (p<0.05 to p<0.0001).

[0103] Figure 14 Quantitative analysis of the relative fluorescence intensity of TAT-NPs, FITC-glucan, and TRITC-glucan in cortical arterioles within 120 minutes after injection in mice.

[0104] Figure 15 Quantitative analysis of the relative fluorescence intensity of the three probes in cortical capillaries within 120 minutes after injection in mice.

[0105] 2. Comparison extended to 4 hours

[0106] In another set of experiments, the imaging time was extended to 4 hours. The results showed that TAT-NPs maintained greater than 90% and greater than 85% of their initial fluorescence intensity in arterioles and capillaries, respectively, after 4 hours, while FITC-glucan and TRITC-glucan decreased to less than approximately 20% and 10%, respectively. Figure 16 , 17 ).

[0107] Figure 16 In vivo fluorescence images of small arteries (left group) and capillaries (right group) in mice (n=4), labeled with TAT-NPs (red channel), FITC-glucan (green channel, commercial probe), or TRITC-glucan (red channel, commercial probe), respectively. Images were acquired at 0, 2, and 4 hours post-injection; scale bar = 50 μm.

[0108] Figure 17 Quantitative analysis of the relative fluorescence intensity of three probes (TAT-NPs, FITC-glucan, and TRITC-glucan) in arterioles (left image) and capillaries (right image) within 0-4 hours after injection in mice.

[0109] Colocalization analysis showed that the fluorescence signals of TAT-NPs and FITC-glucan completely overlapped within the vascular lumen, with no extravascular leakage. Figure 18 , 19 This confirms the high vascular specificity of TAT-NPs.

[0110] Figure 18 and Figure 16 Combined images of TAT-NPs (red channel) and FITC-glucan (green channel) in small arteries (left group) and capillaries (right group) of mice (n=4) at the corresponding imaging time points; scale bar = 50 micrometers.

[0111] Figure 19 Quantitative analysis of the relative fluorescence intensity of TAT-NPs and FITC-glucan in arterioles (left) and capillaries (right) within 0-4 hours after injection in mice.

[0112] Example 6: Evaluation of the photostability of TAT-NPs

[0113] 1. Low-frequency interval imaging

[0114] At a frequency of 1 / 180 Hz (one frame every 3 minutes) for 60 minutes of continuous imaging, the fluorescence intensity of TAT-NPs remained above 95% of the baseline, with no obvious photobleaching observed. Figure 20 , 21 ).

[0115] Figure 20 In vivo two-photon imaging time series of TAT-NPs photobleached mice at a scan frequency of 1 / 180 Hz, with images acquired from baseline before scan to 60 minutes after scan; scale bar = 50 μm.

[0116] Figure 21 The relative fluorescence intensity of TAT-NPs (corresponding to) Figure 20 Quantitative analysis revealed the high photostability at this low scanning frequency.

[0117] 2. High-frequency continuous imaging

[0118] At a frequency of 2.27 Hz for 660 seconds, TAT-NPs maintained clear vascular outlines with only slight fluctuations in fluorescence intensity, indicating good tolerance to high photon flux. Figure 22 , 23 ).

[0119] Figure 22 In vivo two-photon imaging of TAT-NPs photobleached in mice (n=3) at a scanning frequency of 2.27 Hz: the left image shows the full field of view (marked by the dashed box), and the right image is a magnified time-series image of the region (0.5 seconds time interval); scale bar = 50 micrometers.

[0120] Figure 23 The relative fluorescence intensity of TAT-NPs (corresponding to) during the scan period of 0-660 seconds at a frequency of 2.27 Hz. Figure 22 The dynamic changes of the ) reflect the accelerated photobleaching process at high scanning frequencies.

[0121] 3. Long-term retention capability

[0122] Imaging of the same field of view was performed on mice at 0, 2, 4, 8, 24, 48, 72, and 96 hours after injection. Results showed that vascular structures remained clearly discernible at 96 hours, retaining approximately 70% of the initial signal despite a gradual decrease in fluorescence intensity. Figure 24-27 This is sufficient for continuous multi-layered visualization.

[0123] Figure 24 Short-term (0–8 h) and long-term (0–96 h) in vivo two-photon imaging time series of TAT-NPs photobleaching (2.27 Hz scan frequency), showing the evolution of the signal over the extended duration (n=3); scale bar=50 μm.

[0124] Figure 25 Supplementary short-term (0-8 hours) and long-term (0-96 hours) in vivo two-photon imaging sequences of TAT-NPs photobleaching (2.27 Hz scanning frequency) further validated long-term signal retention (n=3); scale bar=50 micrometers.

[0125] Figure 26 correspond Figure 24 Spatial fluorescence intensity distribution: the left image corresponds to short-term (0-8 hours) imaging, and the right image corresponds to long-term (0-96 hours) imaging, showing the temporal variation of signal uniformity.

[0126] Figure 27 correspond Figure 25 Quantitative analysis of relative fluorescence intensity: The left figure corresponds to short-term (0-2.5 hours) imaging, and the right figure corresponds to long-term (0-96 hours) imaging, quantifying the time trend of photobleaching.

[0127] Example 7: Effects of TAT-NPs on Central Nervous System Immune Homeostasis

[0128] 1. Longitudinal imaging to observe microglia morphology

[0129] After injecting TAT-NPs into Cx3cr1-GFP mice, imaging was performed every 5 days for 35 days. Results showed that TAT-NP-labeled blood vessels remained clearly visible, while GFP-labeled microglia maintained a branched, resting morphology with no significant changes in position or morphology. Figure 28 a, 28b).

[0130] Figure 28(a) Longitudinal dual-channel in vivo imaging in mice (n=4): Red channel represents blood vessels labeled with TAT-NPs, and green channel represents microglia / macrophages labeled with Cx3cr1-GFP. Images were acquired continuously from 0 to 35 days post-injection; arrows indicate target microglia / macrophages; scale bar = 50 μm.

[0131] Figure 28 (b) is a magnified view of the green channel (microglia / macrophages) in (a), recorded every 5 days, showing the longitudinal morphological dynamics of individual microglia, scale bar = 10 micrometers.

[0132] 2. Quantitative analysis

[0133] Microglia density remained stable over 35 days, with no significant increase in cell area.

[0134] Figure 29 The quantitative curves of the relative fluorescence intensity of TAT-NPs within 0-35 days after injection reflect the long-term signal retention ability of the probe. Figure 30 The dynamic changes in the number of microglia in each field of view within 0–35 days after injection were characterized to represent the stability of cell density.

[0135] Figure 31 Immunofluorescence staining results of mice (n=3): Iba-1 (green, microglia / macrophage marker), Cd68 (red, lysosomal marker of microglia / macrophage), DAPI (blue, nuclear marker), and merged images of each channel, showing samples from the control group, 1 day after injection of TAT-NPs, and 10 days after injection; scale bar = 50 micrometers.

[0136] Sholl analysis shows that the branch complexity remains unchanged.

[0137] Figure 32 Sholl plots show the curves of the number of intersections of microglia branches as a function of distance from the cell body center, reflecting the differences in morphological complexity among the control group, the 1-day post-injection group, and the 10-day post-injection group. Cells were obtained from three mice in each group (22 cells in the control group, 24 cells in the 1-day group, and 22 cells in the 10-day group).

[0138] Figure 33 The changes in the cell body area of ​​microglia 1 day and 10 days after injection showed no significant change in cell body area.

[0139] Immunofluorescence staining showed that the expression levels of Cd68 (a marker of lysosomal activation) were not significantly different from those in the control group at 1 and 10 days after injection. Figure 34 This indicates that TAT-NPs did not induce microglial cell activation.

[0140] Example 8: TAT-NPs for real-time monitoring of dynamic changes in blood vessel diameter

[0141] In the sensory cortex of mice, continuous imaging was performed at a frame rate of 2.27 Hz while peripheral sensory stimulation (such as tail electrical stimulation) was applied. Multiple vascular segments were selected, and their diameter changes over time were extracted.

[0142] The results showed that TAT-NPs were able to capture the heterogeneous fluctuations such as transient contraction and dilation in different vascular segments. Figure 35 a) After Z-score normalization, the specific responses of each vascular segment can be clearly displayed ( Figure 35 (b) demonstrates the high spatiotemporal resolution of the probe in dynamic monitoring.

[0143] Figure 35 (a) In vivo imaging of sensory cortical vessels labeled with TAT-NPs (red channel): The left group shows the full field of view, with dashed boxes marking four target vascular regions (1-4) from two mice; the right group shows time-series magnified images of each region (0.44-second intervals), with white dashed lines outlining the vessel contours. The full field of view scale bar is 50 micrometers, and the magnified area is 25 micrometers.

[0144] Figure 35 (b) The Z-score dynamic curves corresponding to the four vascular regions in (a) reflect the real-time fluctuations in vascular diameter induced by peripheral input.

[0145] In summary, this invention successfully fabricated a brain vascular two-photon probe, TAT-NPs, based on aggregation-induced emission. This probe exhibits excellent photostability, long-term vascular retention capacity, good biocompatibility, and neuroimmunocompatibility. In a conscious mouse model, TAT-NPs enable multi-scale imaging of the brain vascular network, from acute dynamics to long-term structural remodeling, providing a powerful tool for neurovascular research.

Claims

1. A method for preparing an AIE probe for long-term two-photon imaging of the cerebrovascular system in awake mice, characterized in that, Includes the following steps: (1) Maleic anhydride-triphenylamine and N-(4-aminobutyl)-N-ethyl isoluminol were co-dissolved in the organic solvent N,N-dimethylformamide, and the reaction was carried out in an air atmosphere at 150-170 °C for 6-10 hours using triethylamine catalyst. The resulting reaction solution was first extracted with dichloromethane and then dried with anhydrous magnesium sulfate. The crude product was purified by silica gel column chromatography to obtain the AIE precursor compound. (2) The AIE precursor compound and DSPE-mPEG2000-Mal were co-dissolved in the organic solvent tetrahydrofuran. The resulting mixture was injected into water for ultrasonic emulsification. Then the tetrahydrofuran was removed to obtain AIE nanoparticles with maleimide groups on the surface. (3) The AIE nanoparticles were reacted with the cell-penetrating peptide TAT, coupled by thioether bond, and then purified to obtain TATNPs.

2. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of the AIE precursor compound to DSPE-mPEG2000-Mal is 1:

2.

3. The preparation method according to claim 1, characterized in that, The amino acid sequence of the cell-penetrating peptide TAT in step (3) is RKKRRQRRRC.

4. The preparation method according to claim 1, characterized in that, The purification in step (3) is carried out by ultrafiltration, with the molecular weight cutoff of the ultrafiltration membrane being 100 kDa.

5. An AIE probe obtained by the preparation method according to any one of claims 1-4.

6. The use of the AIE probe of claim 5 in the preparation of a reagent for long-term two-photon imaging of the cerebrovascular system in awake mice.

7. A kit for long-term two-photon imaging of the cerebrovascular system in awake mice, characterized in that, It includes the AIE probe as described in claim 5.

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