Activated near-infrared two-region fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses an activated near-infrared two-region fluorescent probe. The activated near-infrared two-region fluorescent probe comprises a small molecular dye and rare earth nanoparticles, wherein the small molecule dye is an organic small molecule which specifically responds to HClO; the rare earth nanoparticles are of a NaYF4: Yb, Er, Ce-coated NaYF4: Nd core-shell structure, NaYF4: Yb, Er and Ce are taken as a core, and NaYF4: Nd is taken as a shell. According to the invention, the emission of the rare earth nanoparticles at 1550nm is effectively regulated and controlled by utilizing the specific reaction of the small molecule dye and HClO, and the near-infrared two-region fluorescent probe responding to HClO is successfully constructed. The activated near-infrared two-region fluorescent probe is high in signal-to-noise ratio and sensitivity, can be used for in-vitro and in-vivo specific detection of HClO and real-time tracking and monitoring, has the advantages of being large in penetration depth, high in biological safety and the like, further realizes near-infrared two-region biological imaging, can be used for monitoring fluctuation of the concentration of HClO in the brain of an MPTP-induced Parkinson mouse, and can be used for detecting the concentration of the HClO in the brain of the Parkinson mouse. And the treatment effects of various Parkinson's disease treatment drugs are evaluated.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to an activated near-infrared II fluorescent probe, its preparation method, and its application. Background Technology

[0002] Currently, the Parkinson's Disease Rating Scale (PDRS) is used clinically to diagnose Parkinson's disease (PD). However, this is only one means of assessing the condition. Neuroimaging methods are also needed for auxiliary diagnosis to accurately classify patients with different forms of Parkinson's syndrome. In recent years, neuroimaging-based methods have included transcranial ultrasound (TCS), magnetic resonance imaging (MRI), and positron emission tomography (PET). These diagnostic techniques are mainly based on clinical motor symptoms. TCS is limited by penetration depth, operator experience, and resolution, and has low specificity for early diagnosis. MRI imaging is limited by high equipment maintenance costs and long examination times. For PD patients, maintaining a static position for extended periods is difficult due to tremors and movement disorders, thus affecting the accuracy of the results. PET imaging carries certain radiation hazards; the uptake of radioactive tracers can affect the accuracy of the results, and the tracer's lack of specificity limits its clinical ability to differentiate PD from other subtypes. Existing detection methods generally lack sensitivity, making it difficult to accurately capture microscopic brain lesions and accurately diagnose and assess early PD, causing many patients to miss the optimal treatment window.

[0003] Studies have shown that oxidative stress is a core pathogenic mechanism in Parkinson's disease (PD), and HClO, as a strong oxidizing reactive oxygen species (ROS), plays an important role in neuronal damage during PD. Therefore, investigating the molecular regulatory mechanism of HClO on PD is of great significance for elucidating the complex pathogenesis of PD and developing efficient diagnostic and therapeutic methods.

[0004] Many fluorescent probes for detecting HClO are currently used in the detection of various diseases. However, due to the unclear pathogenesis of Parkinson's disease, research in the field of fluorescent probes that can deeply reveal the intrinsic link between HClO and Parkinson's disease from an in vivo perspective using non-destructive detection methods is still very scarce. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an activated near-infrared II fluorescent probe that addresses the shortcomings of the prior art. This probe has high sensitivity and good selectivity and can be used for specific detection of HClO in vitro and in vivo, as well as real-time tracking and monitoring.

[0006] The technical solution adopted by the present invention to solve the above-mentioned problems is as follows:

[0007] An activated near-infrared II fluorescent probe is composed of a small molecule dye and rare earth nanoparticles. The small molecule dye is an organic small molecule that specifically responds to HClO (abbreviated as HBCI-CBO), and its structure is shown in Formula 1. The rare earth nanoparticles are oleic acid-modified NaYF4:Yb,Er,Ce@NaYF4:Nd (abbreviated as OA-RENPs), which form a core-shell structure with NaYF4:Yb,Er,Ce as the core and NaYF4:Nd as the shell. The surface of the core-shell structure is modified with oleic acid ligands.

[0008]

[0009] According to the above scheme, the rare earth nanoparticles have a spherical core-shell structure, with a core diameter of 25–40 nm and a shell thickness generally in the range of 0.9–1.2 nm. The shell thickness is very thin, and the overall size of the spherical core-shell structure is comparable to the core size, generally also in the range of 25–40 nm. Simultaneously, the rare earth nanoparticles contain Nd... 3+ The absorption characteristic peak range is 700–900 nm, which overlaps with the absorption spectrum range of the small molecule dye, which is 600–1200 nm.

[0010] According to the above scheme, the rare earth nanoparticles are synthesized using an oleic acid and octadecene system, with oleic acid ligands on the surface. The molar ratio of NaYF4:Yb,Er,Ce to NaYF4:Nd is 1:(0.15~0.5). By molar percentage, the Ce doping ratio in NaYF4:Yb,Er,Ce is 2%~10%, the Er doping ratio is 0.5%~10%, and the Yb doping ratio is 10%~30%; the Nd doping ratio in NaYF4:Nd is 35%~45%.

[0011] According to the above scheme, the ratio between rare earth nanoparticles and small molecule dyes is 1 mg:(10-85) nmol, preferably 1 mg:(35-85) nmol.

[0012] The preparation method of the above-mentioned activated near-infrared II fluorescent probe includes the following steps:

[0013] (1) Rare earth nanoparticles (OA-RENPs), phospholipid-polyethylene glycol and phospholipid-polyethylene glycol-targeting peptide were dissolved in chloroform, concentrated by nitrogen blowing, and dispersed in water to obtain a solution of rare earth nanoparticles (RENPs@VHP) modified with vascular cell adhesion molecule-1 binding peptide.

[0014] (2) Add a small molecule dye solution to the RENPs@VHP solution, mix and centrifuge to remove the precipitate; the precipitate is the activated near-infrared II fluorescent probe of the present invention, which is dispersed in H3BO3 buffer solution for storage and later use.

[0015] Preferably, in step (1), the phospholipid-polyethylene glycol can be DSPE-PEG; the phospholipid-polyethylene glycol-targeting peptide can be DSPE-PEG-VHPKQHRGGSKGC, where VHPKQHRGGSKGC is a vascular cell adhesion molecule-1 binding peptide. The molecular weight of PEG is preferably 2000–5000 Da.

[0016] According to the above scheme, in step (1), the mass ratio between OA-RENPs and phospholipid-polyethylene glycol and phospholipid-polyethylene glycol-targeting peptide is (60-140):(15-25):1; the dispersion concentration of OA-RENPs in the solvent water is 1-2 mg / mL. In addition, the concentrations of phospholipid-polyethylene glycol and phospholipid-polyethylene glycol-targeting peptide dissolved in chloroform are both 5-15 mg / mL.

[0017] According to the above scheme, in step (2), the small molecule dye solution is obtained by dispersing the small molecule dye in DMF with a concentration of 5-15 mM; when the RENPs@VHP solution is mixed with the small molecule dye solution, the ratio between rare earth nanoparticles and small molecule dye HBCI-CBO is calculated as 1 mg:(10-85) nmol.

[0018] According to the above scheme, in step (2), the pH of the H3BO3 buffer solution is preferably 7.2 to 7.6, and the concentration is preferably 10 to 20 mM.

[0019] Preferably, the present invention provides a method for synthesizing the above-mentioned small molecule dye HBCI-CBO that specifically responds to HClO, comprising the following steps:

[0020] 1) Under a protective atmosphere, 1,8-naphtholactam (compound 1) was dissolved in anhydrous N,N-dimethylformamide (DMF), then NaH was added, and after mixing evenly, the mixture was cooled to -5 to 10°C. Iodohexane was then added, and the mixture was stirred at room temperature for 3 to 4 hours. The mixture was then extracted with ethyl acetate, washed with brine, concentrated, and purified by column chromatography to obtain 1-hexylbenzo[cd]indol-2(1H)-one (compound 2).

[0021] 2) Under an inert gas environment, compound 2 was dissolved in anhydrous THF, and a THF solution of Grignard reagent methyl magnesium iodide (MeMgI) was added dropwise. The mixture was then stirred at 60–70 °C for 1–2 h. After the reaction was completed, the mixture was cooled to room temperature, and hydrochloric acid at around 0 °C was added to quench excess Grignard reagent. This acidic condition also promoted the transfer of the product from the organic phase to the aqueous phase, facilitating separation and purification. After removing THF from the solution by vacuum distillation, KI was added to obtain a red precipitate. The red precipitate was filtered, washed with ethyl acetate and water, and dried to obtain 1-hexyl-2-methylbenzo[cd]indolium iodide (compound 3).

[0022] 3) Compound 3 and squaric acid were mixed in a mixed solvent of n-butanol / toluene and heated to 110-120°C under reflux for 2-3 hours. The solvent in the reaction system was then removed by vacuum distillation, and the crude product was purified by silica gel column chromatography to finally obtain a small molecule dye that specifically responds to HClO.

[0023] The synthetic route of the above-mentioned small molecule dye HBCI-CBO is shown in Formula 2, where 1, 2, and 3 represent compound 1, compound 2, and compound 3, respectively.

[0024]

[0025] According to the above scheme, in step 1), the concentration of 1,8-naphthyllactam in DMF is 0.25-0.5 mol / L; the molar ratio of 1,8-naphthyllactam to iodohexane is 1:(1-1.5), and the molar ratio of 1,8-naphthyllactam to NaH is 1:(2-5); the eluent used for chromatographic purification is composed of petroleum ether (PE) and ethyl acetate (EA) in a volume ratio of (50-10):1.

[0026] According to the above scheme, in step 2), the concentration of compound 2 dissolved in anhydrous THF is 0.2-0.3 mol / L; the molar ratio of compound 2 to methylmagnesium iodide is 1:(1-1.5), the molar ratio of methylmagnesium iodide to hydrochloric acid is 1:(3-4), and the molar ratio of compound 2 to KI is 1:(1-1.5).

[0027] According to the above scheme, in step 3), the molar ratio of compound 3 to squaric acid is (1.5-2.5):1, the mixed solvent is composed of n-butanol and toluene in a volume ratio of 1:(1-1.5), the concentration of compound 3 in the mixed solvent is 0.1-0.2 mol / L, and the eluent used for column chromatography purification is composed of petroleum ether (PE) and ethyl acetate (EA) in a volume ratio of (50-10):1.

[0028] The activated near-infrared II fluorescent probe of the present invention can detect HClO in vitro and in vivo in the near-infrared II region. The application method is as follows: the activated near-infrared II fluorescent probe is added to the test solution, cells or living organisms, and the HClO is dynamically monitored and / or quantitatively analyzed by observing the changes in fluorescence intensity in real time.

[0029] The application of the activated near-infrared II fluorescent probe described in this invention in the non-invasive monitoring of the progression of Parkinson's disease in the brain.

[0030] The technical concept of this invention is as follows:

[0031] This invention, based on the absorption competitive induced emission (ACIE) principle, utilizes the specific reaction between the small molecule dye HBCI-CBO and HClO to achieve effective modulation of the emission of rare earth nanoparticles (NaYF4:Yb,Er,Ce@NaYF4:Nd) at 1550 nm, successfully constructing a near-infrared II (NIR-II) fluorescent probe responsive to HClO. Specifically, at 808 nm, the Nd in the shell of the rare earth nanoparticles... 3+ There is effective spectral overlap between the absorption spectra and those of the small molecule dye HBCI-CBO. Due to the ACIE strategy, the small molecule dye has a higher molar extinction coefficient at 808 nm excitation, and will preferentially absorb the excitation light, thereby reducing the energy of the excitation light directed to Nd. 3+ The transfer of Er in the core of rare earth nanoparticles, thereby quenching the Er 3+ The probe exhibits NIR-II emission at 1550 nm. Upon reaction with HClO, the activated near-infrared II fluorescent probe of this invention disrupts the structure of the small molecule dye HBCI-CBO, affecting the ACIE process and causing a rebound in the NIR-IIb fluorescence signal, which was previously quenched by rare-earth nanoparticles. This enables dynamic monitoring and / or quantitative analysis of HClO. Furthermore, the activated near-infrared II fluorescent probe of this invention is surface-modified with a VHPKQHRGGSKGC peptide targeting the vascular cell adhesion molecule-1 (VCAM-1) receptor on brain endothelial cells, increasing the probe's enrichment in brain tissue and allowing for non-invasive detection of HClO levels in the brain of MPTP-induced Parkinson's disease model mice.

[0032] Compared with the prior art, the beneficial effects of the present invention are:

[0033] (1) The activated near-infrared II fluorescent probe of the present invention has a high signal-to-noise ratio and sensitivity, and can be used for specific detection of HClO in vitro and in vivo as well as real-time tracking and monitoring.

[0034] (2) The activated near-infrared II fluorescent probe of the present invention has the advantages of deep penetration and high biosafety, and further realizes NIR-IIb bioimaging; moreover, it is low in cost and has good selectivity.

[0035] (3) This invention uses NIR-II fluorescence imaging technology to successfully monitor the fluctuation of HClO concentration in the brain of MPTP-induced Parkinson's mice with HClO as the target during oxidative stress, and evaluates the therapeutic effects of various Parkinson's disease treatment drugs. Attached Figure Description

[0036] Figure 1 The following is a schematic diagram illustrating the principle of the activated near-infrared II fluorescent probe for detecting HClO according to the present invention, using an example.

[0037] Figure 2 The 1H NMR spectrum of the organic small molecules used in the examples (the deuterated reagent is DMSO-d6).

[0038] Figure 3 The carbon spectra (with DMSO-d6 as the deuterated reagent) and high-resolution mass spectra of the organic small molecules used in the examples are shown.

[0039] Figure 4 The rare earth nanoparticles Nd in the OA-RENPs in the examples 3+ Absorption and emission spectra of OA-RENPs, HBCI-CBO, and HBCI-CBO-RENPs.

[0040] Figure 5 The images shown are transmission electron microscopy (TEM) images (A core, B core-shell), size distribution histograms (C core, D core-shell), X-ray diffraction patterns (E), and annular dark-field scanning TEM images (F) of the OA-RENPs in the examples.

[0041] Figure 6 Fourier transform infrared spectra of OA-RENPs, RENPs@VHP, HBCI-CBO, and HBCI-CBO-RENPs in Example 1, and RENPs@PEG in the comparative example.

[0042] Figure 7 The Zeta potential diagram (A) and hydrated particle size diagram (B) of RENPs@VHP and HBCI-CBO-RENPs in Example 1 and RENPs@PEG in the comparative example are shown.

[0043] Figure 8 The UV-Vis absorption spectra (A) of HBCI-CBO-RENPs probes with different HBCI-CBO loadings in Example 2, the NIR-IIb fluorescence spectra under 808 nm excitation (B), and the linear relationship between the NIR-IIb fluorescence intensity of HBCI-CBO-RENPs and the HBCI-CBO loading (C) are shown.

[0044] Figure 9The NIR-IIb fluorescence spectra of the probe HBCI-CBO-RENPs reacting with different concentrations of HClO under 808nm excitation in Example 3 are shown in (A); the linear relationship between the NIR-IIb fluorescence intensity of HBCI-CBO-RENPs at 1550nm and the concentration of HClO is shown in (B).

[0045] Figure 10 The specific response of HBCI-CBO-RENPs to HClO in Example 4 (A); the NIR-IIb fluorescence intensity of HBCI-CBO-RENPs at different times in different media at 37℃ (B); the NIR-IIb fluorescence intensity of the probe HBCI-CBO-RENPs after incubation at 37℃ in different pH buffer solutions (C).

[0046] Figure 11 The blood biochemistry and complete blood count levels of mice after intravenous injection of different concentrations of the probe HBCI-CBO-RENPs in Example 5 are as follows: aspartate aminotransferase (AST, U / L), alanine aminotransferase (ALT, U / L), blood urea nitrogen (BUN, mg / dL), creatinine (CREA, μM), urea (UREA, mM), creatine kinase (CK, U / L), and white blood cell count (WBC, 10⁻¹²). 9 / L), red blood cell count (RBC, 10 12 The following parameters were measured: hemoglobin (HGB, g / L), hematocrit (HCT, %), alkaline phosphatase (ALP, U / L), mean corpuscular volume (MCV, fL), mean corpuscular hemoglobin (MCH, pg), and mean corpuscular hemoglobin concentration (MCHC, g / L).

[0047] Figure 12 In Example 5, H&E staining images of major organs of mice after intravenous injection of probes HBCI-CBO-RENPs; scale bar: 200 μm.

[0048] Figure 13 For Example 6, (A) NIR-IIb fluorescence imaging of the brains of mice after intraperitoneal injection of Group a: day 0, Group b: MPTP (20 mg / kg body weight, day 6), Group c: MPTP (20 mg / kg body weight, day 10), and Group d: MPTP (20 mg / kg body weight, day 14), respectively; (B) Normalized NIR-IIb fluorescence intensity of HBCI-CBO-RENPs in the mouse brain under 808 nm laser excitation in Figure (A); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0049] Figure 14NIR-IIb fluorescence imaging of isolated brain tissue from mice injected intraperitoneally with Group a: day 0, Group b: MPTP (20 mg / kg body weight, day 6), Group c: MPTP (20 mg / kg body weight, day 10), and Group d: MPTP (20 mg / kg body weight, day 14) in Example 6; (B) Normalized NIR-IIb fluorescence intensity of isolated brain tissue; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0050] Figure 15 The images show TH immunofluorescence staining after MPTP induction for different days in Example 6 (Group a: 0 days, Group b: 6 days, Group c: 10 days, Group d: 14 days); scale bar: 100 μm.

[0051] Figure 16 Figure 7 shows (A) NIR-IIb imaging of the brains of mice pretreated with different Parkinson's disease drugs (Group a: saline, Group be: levodopa, selegiline, amantadine, piribedil, 20 mg / kg); (B) Normalized NIR-IIb fluorescence intensity of RENPs in the mouse brain under 808 nm laser excitation in Figure (A); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Detailed Implementation

[0052] To more clearly illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0053] In the following examples, the synthesis process of the small molecule dye HBCI-CBO specifically includes the following steps:

[0054] (1) Under an inert gas environment, 1,8-naphtholactam (846 mg, 5 mmol) was dissolved in 15 mL of anhydrous DMF, and then NaH (360 mg, 15 mmol) was added. The resulting mixture was cooled to 0 °C, and then iodohexane (1.27 g, 6 mmol) was added. The mixture was then stirred at room temperature for 3 h. After the reaction was completed, the mixture was extracted with ethyl acetate, washed with brine and concentrated, and then purified by column chromatography (the eluent consisted of PE and EA in a volume ratio of 20:1) to obtain compound 2.

[0055] (3) Under an inert gas environment, compound 2 (1.26 g, 5 mmol) was dissolved in 20 mL of anhydrous THF, and 2 mL of MeMgI solution (3 mol / L, THF solvent) was added dropwise over 10 minutes. The reaction was stirred at 60 °C for 1 h. After the reaction was completed, the mixture was cooled to room temperature, and the resulting mixture was slowly poured into 10 mL of 2 mol / L HCl (ice-water bath) to quench excess Grignard reagent and to bring the system to acidic conditions to promote the transfer of the product from the organic phase to the aqueous phase. After removing THF from the system by vacuum distillation, KI (1 mol / L, 5 mL) solution was added to obtain a red precipitate. The red precipitate was filtered, washed with ethyl acetate and water, and dried to obtain compound 3.

[0056] (3) Compound 3 (0.6 g, 1.6 mmol) was mixed with squaric acid (0.1 g, 0.8 mmol) and refluxed at 110–120 °C for 2 h in a mixture of n-butanol / toluene (total volume 10 mL, volume ratio 1:1). The solvent was then removed under reduced pressure. The resulting solid was purified by silica gel column chromatography (eluting agent composed of PE and EA in a volume ratio of 20:1) to obtain a small molecule dye with the structure shown in Formula 1. Its characterization was performed by 1H NMR, 1C NMR, and high-resolution mass spectrometry. The results are as follows:

[0057] HBCI-CBO NMR test data: 1 H NMR (400MHz, CDCl3) δ9.20(s,2H),7.94(d,J=8.0Hz,2H),7.84(t,J=7.7Hz,2H),7.56(d,J=8.2Hz,2H),7.52–7.47(m,3H),7.05(d ,J=7.1Hz,2H),6.39(s,2H),4.18(s,4H),1.90(p,J=7.7Hz,6H),1.49–1.44(m,6H),1.34(d,J=3.3Hz,4H),0.88(d,J=7.1Hz,6H). like Figure 2 As shown.

[0058] 13C NMR (101MHz, CDCl3) δ166.74,150.09,140.69,131.54,129.86,129.55,128.78,128.7 5,128.54,128.16,127.82,127.39,124.58,120.58,106.00,91.14,76.36,76.04,75.7 2,67.20,52.40,43.08,37.81,30.72,30.58,30.55,29.41,28.68,28.33,28.13,27.95,27.90,27.01,26.04,25.82,22.81,21.97,21.63,21.53,17.49,13.00,12.97,9.95. (For example...) Figure 3 As shown.

[0059] HRMS-ESI(m / z):calcd.For C 40 H 40 N₂O₂ 581.3246 [M+H] + ; found 581.3162[M+H] + .like Figure 4 As shown.

[0060] The preparation method of rare earth nanoparticles OA-RENPs used in the following embodiments is as follows:

[0061] (1) Synthesis of rare earth oleate (RE(oleate)3): First, 10 mmol of rare earth halide RECl3 (RE selected from Y, Yb, Er, Ce, Nd, respectively, to synthesize the corresponding rare earth oleate) and 30 mmol of sodium oleate solid were added to a mixed solvent consisting of 15 mL of high-purity water, 20 mL of ethanol, and 35 mL of hexane. The mixture was then placed in a 250 mL round-bottom flask and refluxed at 70 °C for 6 h. After the reaction was complete, the mixture was separated and washed with water and ethanol. The solution was then concentrated under reduced pressure, and oleic acid and octadecene were added to dissolve and quantify the solution, finally yielding a RE(oleate)3 (RE = Y, Yb, Er, Ce, Nd) solution with an ion concentration of 0.25 mol / L.

[0062] (2) Synthesis of oleic acid modified NaYF4:Yb,Er,Ce@NaYF4:Nd(OA-RENPs): First, according to the molar ratio of core composition Y(oleate)3:Yb(oleate)3:Er(oleate)3:Ce(oleate)3 of 73:20:2:5, the corresponding RE(oleate)3 solutions prepared in step (1) were mixed to obtain the core RE(oleate)3 solution, and the total RE concentration was still 0.25mol / L; according to the molar ratio of shell composition Y(oleate)3:Nd(oleate)3 of 60:40, the corresponding RE(oleate)3 solutions prepared in step (1) were mixed, and then mixed with oleic acid and octadecene at a volume ratio of 1:2:2 to obtain the shell RE(oleate)3 solution, and the total RE concentration was 0.05mol / L.

[0063] Next, oleic acid and 1-octadecene (volume ratio 1:1) were pre-filled into a 100 mL three-necked flask as a solvent. Then, a core RE (oleate) 3 solution with a total RE content of 1 mmol and 20 mmol of sodium fluoride were added sequentially, resulting in a total mixture volume of 20 mL. This system was then subjected to an argon atmosphere, and the temperature was raised to 110 °C for 1 h to remove water and oxygen. The temperature was then raised to 290 °C for 2 h. Next, 5 mL of shell RE (oleate) 3 solution was injected using a microsyringe, and the reaction was continued at 290 °C for 1 h. After the reaction was complete, the mixture was allowed to cool naturally to room temperature, and the precipitate obtained after centrifugation was collected; this was OA-RENPs. The precipitate was washed three times with n-hexane, and ethanol was added to precipitate clean OA-RENPs. These OA-RENPs could be dispersed in n-hexane, and their concentration was quantified for later use.

[0064] like Figure 5 It can be seen that the OA-RENPs prepared by the above method are spherical with uniform size, the diameter of the core is 28.6±1.2nm, the diameter of the core-shell structure is 30.9±1.0nm, and the shell thickness is about 1.2nm; in addition, the crystal form of both the core and the core-shell RENPs is hexagonal, which is consistent with the standard card of β-NaYF4.

[0065] Comparative Example

[0066] OA-RENPs and DSPE-PEG 2000 Dissolve the PEG in chloroform at a mass ratio of 5:1. Dry the resulting mixture with nitrogen gas. Then disperse the residue (abbreviated as RENPs@PEG) in ultrapure water and centrifuge at 13300 rpm for 15 min. Discard any residue containing excess DSPE-PEG. 2000The supernatant was used to wash the resulting precipitate twice with ultrapure water, and then resuspended in 10 mM H3BO3 buffer solution (pH = 7.4) to disperse it into a RENPs@PEG solution with a concentration of 1 mg / mL, which was compared with Example 1.

[0067] Example 1

[0068] A method for preparing an activated near-infrared II fluorescent probe includes the following steps:

[0069] DSPE-PEG 2000 (0.95mg) and DSPE-PEG 2000 -VHPKQHRGGSKGC (0.05 mg) was dissolved in chloroform at a concentration of 10 mg / mL and added to OA-RENPs (5 mg). The resulting mixture was dried under nitrogen. The residue (abbreviated as RENPs@VHP) was then dispersed in 1 mL of ultrapure water at a rare earth nanoparticle concentration of 1 mg / mL. 8.5 μL of a 10 mM DMF solution of small molecule dye was slowly added by vortexing. The mixture was then centrifuged at 13300 rpm for 15 min, and excess DSPE-PEG was discarded. 2000 -VHPKQHRGGSKGC、DSPE-PEG 2000 The supernatant of HBCI-CBO was used to obtain the precipitate, which is the activated near-infrared II fluorescent probe (HBCI-CBO-RENPs). After washing twice with ultrapure water, it was resuspended in 10 mM H3BO3 buffer solution (pH=7.4) and dispersed into a 1 mg / mL HBCI-CBO-RENPs solution for later use.

[0070] like Figure 6 As shown, HBCI-CBO-RENPs are located at 1741 cm⁻¹. -1 and 1643cm -1 The stretching vibration peak at that location, and DSPE-PEG 2000 The matching stretching vibrations of the amide bond and the carbon-oxygen double bond (C=O) of the ester group confirm the existence of DSPE-PEG. 2000 Successfully coated on the surface of RENPs; furthermore, HBCI-CBO-RENPs at 1281 cm⁻¹ -1 and 1240cm -1 The stretching vibration peak at that location, and DSPE-PEG 2000 - The C-N bond stretching vibrations in the peptide bonds of VHP are consistent, which confirms that DSPE-PEG 2000 -VHP has been successfully coated onto the RENPs surface. HBCI-CBO-RENPs are located at 1563 cm⁻¹. -1The stretching vibration peak at the location corresponds to the stretching vibration peak of the benzene ring skeleton in the naphthalene ring structure of HBCI-CBO, indicating that the small molecule dye HBCI-CBO was successfully loaded.

[0071] Since HBCI-CBO is nearly neutral in aqueous solution, the Zeta potential of RENPs@VHP after assembly with HBCI-CBO changes from -8.9 mV for RENPs@VHP to -7.8 mV for HBCI-CBO-RENPs. Figure 7 A) indicates that HBCI-CBO was successfully encapsulated in the hydrophobic cavity; by Figure 7 As can be seen from B, after coating, the hydrated particle size of the probe HBCI-CBO-RENPs also increases, and its hydrated particle size is 105nm.

[0072] The above test results prove that the fluorescent probes HBCI-CBO-RENPs were successfully constructed in this embodiment.

[0073] Example 2

[0074] The difference between Example 2 and Example 1 lies in the loading amount of small molecule dye on the surface of rare earth nanoparticles. In Example 2, the ratios of small molecule dye to rare earth nanoparticles were 0 nmol:1 mg, 12.4 nmol:1 mg, 16.6 nmol:1 mg, 20.9 nmol:1 mg, 21.1 nmol:1 mg, 37.2 nmol:1 mg, 45.3 nmol:1 mg, 51.1 nmol:1 mg, and 85 nmol:1 mg. When the small molecule dye content was 0, it served as a blank control.

[0075] The absorption spectra of HBCI-CBO-RENPs probes with different HBCI-CBO loadings prepared in Example 2 are as follows: Figure 8 As shown in Figure A, when 85 nmol HBCI-CBO is loaded onto 1 mg of RENPs, the absorbance of the probe HBCI-CBO-RENPs reaches its peak at 808 nm; as shown in Figure A. Figure 8 As shown in B and C, under 808 nm excitation, the fluorescence signal of HBCI-CBO-RENPs at 1550 nm gradually weakens with increasing loading of the small molecule dye HBCI-CBO. When 1 mg of RENPs is loaded with 85 nmol of HBCI-CBO, the fluorescence intensity at 1550 nm is quenched by 93%, indicating that HBCI-CBO has a good quenching effect on rare earth nanoparticles. Subsequent examples all selected HBCI-CBO-RENPs (i.e., the fluorescent probe prepared in Example 1) with a loading of 85 nmol / mg to test its optical properties and biological applications.

[0076] Example 3

[0077] The application of activated near-infrared II fluorescent probes in the near-infrared II region for the detection of HClO is as follows: Add 0–200 μM HClO sequentially to H3BO3 buffer (pH = 7.4, 20 mM) (specific concentrations are shown in the image). Figure 9 After adding probe HBCI-CBO-RENPs at a concentration of 1 mg / ml and incubating for 15 min, the NIR-IIb fluorescence signal of probe HBCI-CBO-RENPs at 1550 nm was detected under 808 nm excitation.

[0078] Test results as follows Figure 9 As shown, the ACIE process was disrupted after HBCI-CBO-RENPs reacted with different concentrations of HClO, and the NIR-IIb fluorescence signal increased with increasing HClO concentration. A linear relationship was fitted between the NIR-IIb fluorescence intensity at 1550 nm and the HClO concentration, revealing a good linear relationship. The linear equation was y = 0.004c + 0.2054 (where c is the HClO concentration and y is the fluorescence intensity).

[0079] Example 4

[0080] To evaluate the specific response of the probe HBCI-CBO-RENPs to the target analyte HClO, an interference experiment was conducted using the interfering agent GSH (500 μM). 1 O2(500μM), Cys(500μM), H2O2(500μM), Hcy(500μM), ONOO - (500μM), O2 ·- 500 μM HClO, ·OH (500 μM), and H2S (500 μM) were used to replace HClO in Example 3, and the NIR-IIb fluorescence intensity at 1550 nm was detected. Other experimental conditions were the same as in Example 3. 1 O2 is generated in situ by adding HClO to an excess H2O2 solution (ClO2). - / H2O2=1:5); ·OH is generated in situ by adding FeCl2 to excess H2O2 solution (Fe 2+ / H2O2=1:6); ONOO - The solution is generated in situ by adding HCl to a solution of NaNO2 and excess H2O2 at 0°C, followed by the rapid addition of NaOH (HCl / NaNO2 / H2O2 / NaOH = 6:6:7:3); O2 ·- It is prepared by dissolving a certain amount of KO2 powder in dimethyl sulfoxide.

[0081] like Figure 10As shown in Figure A, when common interfering molecules such as ROS, RNS, and reactive thiols are present in the system, the fluorescence intensity of the probe HBCI-CBO-RENPs at 1550 nm does not fluctuate significantly under 808 nm excitation. Only when HClO is present in the system does the probe exhibit a strong fluorescence response, demonstrating high selectivity. This result fully demonstrates that HBCI-CBO-RENPs have excellent specific recognition ability for HClO, and in a biological environment, can effectively eliminate the influence of other interfering substances and accurately detect HClO.

[0082] Stability test of the probe: The probe HBCI-CBO-RENPs was dispersed at a concentration of 1 mg / ml in different media (H3BO3 pH=7.4, 20 mM, DMEM basal medium (containing 4.5 g / L L-glucose), fetal bovine serum (10% FBS), whole blood), and incubated in a shaker at 37℃. The NIR-IIb fluorescence signal of the probe HBCI-CBO-RENPs at 1550 nm was detected under 808 nm excitation. The NIR-IIb fluorescence intensity remained basically stable within 24 h. Figure 10 B).

[0083] Furthermore, incubating the probe HBCI-CBO-RENPs in H3BO3 buffer at different pH values ​​for 2 hours did not significantly change the NIR-IIb fluorescence intensity of the probe HBCI-CBO-RENPs. Figure 10 C).

[0084] The above results demonstrate that the probe HBCI-CBO-RENPs exhibits specific response to the target HClO and good stability, making it suitable for in vivo imaging.

[0085] Example 5

[0086] To investigate the biosafety of the activated near-infrared II fluorescent probe, healthy C57BL / 6 mice were injected via tail vein into the experimental group (HBCI-CBO-RENPs 15 mg / 100 g body weight). The control group consisted of mice that did not receive the probe. Blood routine analysis and serum biochemical analysis were performed on the mice in each group at 7 and 14 days later. Figure 11 As shown, the major organs (heart, liver, spleen, lungs, kidneys, and brain) were stained with H&E and then observed and photographed under a microscope.

[0087] Depend on Figure 11 It can be seen that there were no significant differences in the various indicators of routine blood tests and blood biochemistry in mice; Figure 12H&E staining results also confirmed that the probe HBCI-CBO-RENPs did not cause significant damage to the major organs of mice. These results indicate that the probe HBCI-CBO-RENPs has good biocompatibility.

[0088] Example 6

[0089] Application of the probe HBCI-CBO-RENPs in non-invasive monitoring of Parkinson's disease progression in the mouse brain: MPTP is a neurotoxin that selectively destroys dopaminergic neurons to induce Parkinson's disease, thereby upregulating MPO expression and HClO levels. Mouse models of Parkinson's disease with different severities were established by intraperitoneal injection of MPTP for different durations (20 mg / kg, Group a: day 0, Group b: day 6, Group c: day 10, Group d: day 14). Then, the probe HBCI-CBO-RENPs was injected via the tail vein. Ten hours later, the brains of the Parkinson's disease model mice were examined. NIR-IIb fluorescence imaging showed that the NIR-IIb fluorescence intensity in the mouse brain increased with the increase of MPTP induction days (e.g., ...). Figure 13 (As shown in A).

[0090] When the MPTP induction period was 14 days, the NIR-IIb luminescence intensity in the mouse brain was significantly higher than that in Group a, indicating that MPTP-induced increase in HClO production in the mouse brain ( Figure 13 B), and closely related to the degree of damage, these NIR-IIb imaging results demonstrate that the probe HBCI-CBO-RENPs can detect changes in endogenous HClO content caused by MPTP stimulation in mice.

[0091] Mice from Group a to Group d were sacrificed at 10 h, and brain tissue was isolated and imaged in vitro. Figure 14 The results (A and B) further demonstrate that the probe HBCI-CBO-RENPs can detect changes in endogenous HClO levels in the brains of MPTP-induced Parkinson's disease model mice in vivo.

[0092] Simultaneously, immunofluorescence staining of the substantia nigra region of isolated brain tissue for tyrosine hydroxylase (TH) was performed, such as... Figure 15 As shown, the loss of TH-positive cells in dopaminergic neurons in the substantia nigra region was more severe with the increase of MPTP induction days, further confirming that different degrees of Parkinson's disease were induced by intraperitoneal injection of MPTP for different numbers of days, and the results of TH immunofluorescence staining were consistent with the results of NIR-IIb fluorescence imaging using probes HBCI-CBO-RENPs in vivo.

[0093] Example 7

[0094] To explore the feasibility of using the probe HBCI-CBO-RENPs for in vivo efficacy evaluation: C57BL / 6 male mice were randomly divided into five groups (n=3 per group). Each group underwent intraperitoneal injection. Group a served as the control group, receiving 20 mg / kg saline. Groups b through e received levodopa, selegiline, amantadine, and piribedil, respectively, at 20 mg / kg. All groups received intraperitoneal injections for 7 consecutive days. Subsequently, HBCI-CBO-RENPs (75 μL, 40 mg / mL) were injected via the tail vein. NIR-IIb imaging was acquired for 10 hours under 808 nm laser excitation. Figure 16 As shown in Figures A and B, compared to Group a, the NIR-IIb fluorescence signals of the treatment groups receiving levodopa, selegiline, amantadine, and piribedil were reduced to varying degrees. These results indicate that HClO levels were reduced in the brains of mice treated with these anti-Parkinson's drugs, revealing that these four drugs have different therapeutic effects on Parkinson's disease. The probe HBCI-CBO-RENPs can monitor HClO levels in the in vivo brain via fluorescence imaging and assess the efficacy of anti-Parkinson's drugs.

[0095] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. An activated near-infrared II fluorescent probe, characterized in that, It includes small molecule dyes and rare earth nanoparticles; wherein, the small molecule dyes are organic small molecules that specifically respond to HClO, and the structure is shown in Formula 1; the rare earth nanoparticles are NaYF4:Yb,Er,Ce@NaYF4:Nd core-shell structures, with NaYF4:Yb,Er,Ce as the core and NaYF4:Nd as the shell.

2. The activated near-infrared II fluorescent probe according to claim 1, characterized in that, The rare earth nanoparticles have a spherical core-shell structure, with a core size of 25–40 nm and a shell thickness of 0.9–1.2 nm; the rare earth nanoparticles contain Nd 3+ The absorption characteristic peak range is 700–900 nm, which overlaps with the absorption spectrum range of the small molecule dye, which is 600–1200 nm.

3. The activated near-infrared II fluorescent probe according to claim 1, characterized in that, The rare earth nanoparticles are synthesized using an oleic acid and octadecene system, with oleic acid ligands on their surface. The molar ratio of NaYF4:Yb,Er,Ce to NaYF4:Nd is 1:(0.15–0.5). By molar percentage, the Ce doping ratio in NaYF4:Yb,Er,Ce is 2%–10%, the Er doping ratio is 0.5%–10%, the Yb doping ratio is 10%–30%, and the Nd doping ratio in NaYF4:Nd is 35%–45%.

4. The activated near-infrared II fluorescent probe according to claim 1, characterized in that, The ratio between rare earth nanoparticles and small molecule dyes is 1 mg:(10-85) nmol.

5. The method for preparing an activated near-infrared II fluorescent probe according to claim 1, characterized in that, Includes the following steps: (1) Rare earth nanoparticles, phospholipid-polyethylene glycol and phospholipid-polyethylene glycol-targeting peptides were dissolved in chloroform, concentrated by nitrogen blowing, and then dispersed in water to obtain RENPs@VHP solution. (2) Add a small molecule dye solution to the RENPs@VHP solution, mix and centrifuge to remove the precipitate; the precipitate is the activated near-infrared II fluorescent probe.

6. The method for preparing an activated near-infrared II fluorescent probe according to claim 5, characterized in that, In step (1), the mass ratio between rare earth nanoparticles and phospholipid-polyethylene glycol and phospholipid-polyethylene glycol-targeting peptide is (60-140):(15-25):1; the dispersion concentration of rare earth nanoparticles in the solvent water is 1-2 mg / mL.

7. The method for preparing an activated near-infrared II fluorescent probe according to claim 5, characterized in that, In step (2), the small molecule dye solution is obtained by dispersing the small molecule dye in DMF with a concentration of 5-15 mM; when the RENPs@VHP solution is mixed with the small molecule dye solution, the ratio between rare earth nanoparticles and small molecule dye is calculated as 1 mg:(10-85) nmol.

8. The method for preparing an activated near-infrared II fluorescent probe according to claim 5, characterized in that, The synthesis method of small molecule dyes includes the following steps: 1) Under a protective atmosphere, 1,8-naphtholactam was dissolved in DMF, then NaH was added, and after mixing evenly, the mixture was cooled to -5 to 10°C. Iodohexane was then added, and the mixture was stirred at room temperature for 3 to 4 hours. The mixture was then extracted with ethyl acetate, washed with brine, concentrated, and purified by column chromatography to obtain 1-hexylbenzo[cd]indol-2(1H)-one. 2) Under an inert gas environment, 1-hexylbenzo[cd]indol-2(1H)-one was dissolved in anhydrous THF, and Grignard reagent methyl magnesium iodide was added dropwise. The reaction was stirred at 60-70℃ for 1-2 h. After the reaction was completed, the mixture was cooled to room temperature, and hydrochloric acid was added to quench the Grignard reagent. Then, THF was removed by vacuum distillation, and KI was added to obtain a red precipitate. After washing and drying, 1-hexyl-2-methylbenzo[cd]indolium iodide was obtained. 3) Mix 1-hexyl-2-methylbenzo[cd]indolium iodide and squaric acid in a mixed solvent of n-butanol / toluene, heat to 110-120°C and reflux for 2-3 hours, then remove the solvent from the reaction system by vacuum distillation, and then purify by silica gel column chromatography to obtain the small molecule dye.

9. The method for preparing an activated near-infrared II fluorescent probe according to claim 8, characterized in that, In step 1), the molar ratio of 1,8-naphtholactam to iodohexane is 1:(1-1.5), and the molar ratio of 1,8-naphtholactam to NaH is 1:(2-5). In step 2), the molar ratio of 1-hexylbenzo[cd]indol-2(1H)-one to methylmagnesium iodide is 1:(1-1.5), and the molar ratio of 1-hexylbenzo[cd]indol-2(1H)-one to KI is 1:(1-1.5). In step 3), the molar ratio of 1-hexyl-2-methylbenzo[cd]indolium iodide to squaric acid is (1.5–2.5):

1.

10. The application of the activated near-infrared II fluorescent probe of claim 1 in detecting HClO or in non-invasive monitoring of the progression of Parkinson's disease in the brain.

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