BODIPY fluorescent dye, preparation method and application thereof, and FRET-ACQ double-effect near-infrared probe

Abstract

The invention relates to a BODIPY fluorescent dye, a preparation method and application thereof and an FRET-ACQ double-effect near-infrared probe, and provides the BODIPY fluorescent dye with the structural formula shown in the formula 1. Wherein R is alkyl with 1-5 carbons. The BODIPY fluorescent dye has the beneficial effects that the BODIPY fluorescent dye (BDP1) is a probe with bright NIR-I region fluorescence and excellent ACQ property, so that the BODIPY fluorescent dye (BDP1) can be independently used as an ACQ probe; meanwhile, the absorption and emission wavelengths of the BDP1 are matched with those of the ACQ probe P1, and possibility is provided for combined use of the BDP1 and the ACQ probe P1. The method can improve the tracing accuracy of a drug carrier in a living body, facilitates clarification of the in-vivo action mechanism of the nano-drug, optimizes the nano-drug in a targeted manner, and has great clinical transformation significance.

Description

Technical Field

[0001] This invention relates to the field of organic fluorescent dye technology, and in particular to BODIPY fluorescent dye, its preparation method and application, and FRET-ACQ dual-effect near-infrared probe. Background Technology

[0002] Nanomedicines, delivered precisely to lesion sites via specific nanocarriers, enhance efficacy and reduce toxicity, demonstrating promising applications in the treatment of various diseases, including cancer, infectious diseases, and neurological disorders. However, due to limited understanding of their in vivo behavior, the clinical translation rate of nanomedicines is very low. Nanomedicines consist of three main components in vivo: drug-loaded particles, the active drug, and the carrier material. The relationships between these components and their interaction with the body are constantly evolving. Real-time monitoring of the in vivo fate of drug-loaded particles (i.e., nanocarriers) provides crucial information on their biodistribution, interactions with the body, drug release, biodegradation, metabolism, and elimination, thus elucidating the in vivo mechanisms of action and enabling targeted optimization of nanomedicines, which has significant clinical translational implications.

[0003] Due to the small size of nanocarriers and their constant dynamic degradation process, real-time, in vivo monitoring of nanocarriers is extremely difficult. To effectively distinguish between nanocarriers and free probes and avoid interference from free probes, environmentally responsive fluorescent probes have been developed, such as a series of probes based on aggregation-induced emission (AIE), fluorescence resonance energy transfer (FRET), and aggregation-induced quenching (ACQ) mechanisms, which can differentiate the signals of nanocarriers and free probes to a certain extent.

[0004] However, AIE probes tend to re-aggregate and emit light after being released from nanocarriers, and their wavelengths are typically short, limiting their application in in vivo imaging. FRET probes refer to the energy transfer phenomenon from donor to acceptor when the donor emission spectrum and acceptor absorption spectrum highly overlap and the distance between the donor and acceptor is less than 10 nm. When nanocarriers are labeled with FRET probes, the small distance between the donor and acceptor leads to the FRET effect, resulting in weakened donor fluorescence and bright acceptor fluorescence (FRET signal). When the nanocarrier degrades or deaggregates, the donor and acceptor separate, the distance increases, the FRET effect disappears, donor fluorescence recovers, and acceptor fluorescence disappears. Monitoring the FRET signal or the ratio of FRET to donor signal allows for the overall identification of the nanocarrier. Although FRET probes have been used for in vivo monitoring of various nanomedicine carriers, free FRET probes dispersed in single-molecule form often easily re-accumulate in cell membranes or organs, producing recurring signals and even causing uninterpretable results.

[0005] The applicant has taken a different approach, developing several environmentally responsive BODIPY near-infrared I / II fluorescent probes (P1, P2, FD-B21, FD-C7, and ACQ1) based on the ACQ effect. These probes are dispersed as single molecules within the lipophilic core of a nanocarrier, emitting strong near-infrared fluorescence (ON). When the nanocarrier degrades or depolymerizes, the probes are released into the surrounding water environment, where they immediately aggregate based on π-π interactions, resulting in rapid and complete fluorescence quenching (OFF). This effectively overcomes the interference of free probes on the nanocarrier, enabling accurate real-time tracking within the nanocarrier. However, aggregated and quenched free ACQ probes can redisperse in the lipophilic environment of biomolecules or phospholipids, causing fluorescence re-emergence and some interference, thus reducing the accuracy of in vivo monitoring and analysis. Summary of the Invention

[0006] (a) Technical problems to be solved

[0007] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides BODIPY fluorescent dye and its preparation method and application, and FRET-ACQ dual-effect near-infrared probe, which solves the technical problem that the accuracy of in vivo monitoring of existing drug-loaded particles (nanocarriers) needs to be further improved.

[0008] (II) Technical Solution

[0009] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0010] In a first aspect, embodiments of the present invention provide a BODIPY fluorescent dye having the structural formula shown in Formula 1:

[0011] ;

[0012] In this structure, R is an alkyl group with 1-5 carbons, the 1 and 7 positions are phenyl groups, the 3 and 5 positions are p-alkoxyphenyl groups, and a six-membered fused ring structure is formed on both sides.

[0013] Secondly, embodiments of the present invention provide a method for preparing the aforementioned BODIPY fluorescent dye, comprising the following steps:

[0014] S1. In an ice-water bath, phosphorus oxychloride is added dropwise to an equimolar amount of N,N-dimethylformamide. After stirring for 10-15 minutes, the mixture is brought to room temperature and stirred for another 15-20 minutes to obtain the first reaction solution.

[0015] S2. Add a 1,2-dichloroethane solution of 7-alkoxy-4,5-dihydro-1H-benzo[g]indole-3-phenyl to the first reaction solution to carry out the Vilsmeier reaction, and synthesize and isolate the product 7-alkoxy-4,5-dihydro-1H-benzo[g]indole-3-phenyl-2-carboxaldehyde.

[0016] S3. Under an inert gas atmosphere and at room temperature, 7-alkoxy-4,5-dihydro-1H-benzo[g]indole-3-phenyl-2-carboxaldehyde and an equimolar amount of 7-alkoxy-4,5-dihydro-1H-benzo[g]indole-3-phenyl are dissolved in redistilled and dried 1,2-dichloroethane. Phosphorus oxychloride is then added dropwise, and the mixture is stirred until homogeneous. After reacting for 12-14 hours, triethylamine and boron trifluoride diethyl ether are added, and the mixture is stirred at room temperature for 4-5 hours to obtain the second reaction solution. The alkoxy group is an alkoxy group with 1-5 carbon atoms.

[0017] S4. The second reaction solution was concentrated and subjected to column chromatography to separate a green metallic solid, which was the BODIPY fluorescent dye shown in Formula 1.

[0018] Thirdly, embodiments of the present invention provide an application of the aforementioned BODIPY fluorescent dye in fluorescent tracing of nanocarriers in living organisms.

[0019] In a preferred embodiment of the present invention, the application uses BODIPY fluorescent dye as a donor ACQ probe and P1 as an acceptor ACQ probe. The BODIPY fluorescent dye and P1 together form a FRET-ACQ dual-effect near-infrared probe, which is used for fluorescence tracing of nanocarriers in vivo. The structural formula of P1 is shown in Formula 2.

[0020] .

[0021] P1 is an existing compound; see Nanomedicine 2015, 11 (8), 1939-1948 for details.

[0022] As a preferred embodiment of the present invention, in the application described, when the FRET-ACQ dual-effect near-infrared probe is loaded on a nanocarrier, the concentration of BODIPY fluorescent dye used is 10-40 μM, and the concentration of P1 used is 5-15 μM.

[0023] As a preferred embodiment of the present invention, the application described herein involves the FRET-ACQ dual-effect near-infrared probe being encapsulated within a nanocarrier.

[0024] The nanocarrier has a hydrophobic matrix structure or a hydrophobic core-hydrophilic shell structure;

[0025] The FRET-ACQ dual-effect near-infrared probe is encapsulated in a hydrophobic matrix or hydrophobic core of a nanocarrier.

[0026] As a preferred embodiment of the present invention, in the application described herein, the hydrophobic matrix structure is at least one of polymer nanoparticles / microspheres, solid lipid microparticles / nanoparticles, microemulsions, nanoemulsions, and nanocrystals.

[0027] The hydrophobic core-hydrophilic shell structure is a polymer nanomicelle.

[0028] In a preferred embodiment of the present invention, the nanocarrier is at least one of polymeric micelles, polymeric nanoparticles, solid lipid nanoparticles, nanoemulsions, inorganic nanoparticles, nanoemulsions, and nanocrystals.

[0029] In a preferred embodiment of the present invention, the nanocarrier is a nanoscale material used for drug delivery or gene delivery.

[0030] Thirdly, embodiments of the present invention provide a FRET-ACQ dual-effect near-infrared probe, which includes: a donor ACQ probe and an acceptor ACQ probe, wherein the acceptor ACQ probe is a BODIPY fluorescent dye with the structure shown in Formula 1, and the acceptor ACQ probe is a compound with the structure shown in Formula 2.

[0031] Equations 1 and 2 are shown below:

[0032] , .

[0033] (III) Beneficial Effects

[0034] The beneficial effects of this invention are as follows: The BODIPY fluorescent dye, its preparation method, and its application, along with the FRET-ACQ dual-effect near-infrared probe, utilize the fact that BODIPY fluorescent dye (BDP1) is a probe with bright fluorescence in the NIR-I region and excellent ACQ properties. It can be used alone as an ACQ probe; simultaneously, the absorption and emission wavelengths of BDP1 match those of the ACQ probe P1, making combined use possible. The two can form the FRET-ACQ dual-effect probe. Compared to using BDP1 or P1 alone, this FRET-ACQ dual-effect probe significantly reduces interference from fluorescence re-enhancing after carrier disintegration when used to label in vivo drug carriers, demonstrating a clear advantage in the accuracy of drug carrier tracking in vivo. Compared to existing technologies, it can improve the accuracy of drug carrier tracking in vivo, facilitating the elucidation of the in vivo mechanism of action of nanomedicines and enabling targeted optimization of nanomedicines, thus possessing significant clinical translational value. Attached Figure Description

[0035] Figure 1The graph shows the fluorescence intensity at the maximum emission wavelength of BDP1 and P1 in acetonitrile / water mixed solvents with different water contents as a function of water content.

[0036] Figure 2 Normalized absorption (solid line) and emission (dashed line) of BDP1 and P1 in chloroform.

[0037] Figure 3 Absorption spectra of PM after 10-fold dilution with BDP1 (10 μM, A; 20 μM, B; 30 μM, C; 40 μM, D) and P1 (5 μM, 10 μM and 15 μM) co-labeled or separately labeled;

[0038] Figure 4 Fluorescence spectra of PM co-labeled or individually labeled with BDP1 (10 μM, A; 20 μM, B; 30 μM, C; 40 μM, D) and P1 (5 μM, 10 μM and 15 μM) (λex = 642 nm); insets show magnified fluorescence spectra of each in the 700-850 nm range.

[0039] Figure 5 The fluorescence intensity at 749 nm is calculated for PM co-labeled with different concentrations of BDP1 and P1 and the corresponding single-probe labeled PM; the inserted text indicates the factor by which the fluorescence intensity of the co-labeled PM increases relative to the BDP1 label;

[0040] Figure 6 The fluorescence reproduction of dissolved FRET-ACQ probes (BDP1+P1), BDP1, and P1 in plasma;

[0041] Figure 7 Fluorescence reproduction of pre-quenched FRET-ACQ probes (BDP1+P1), BDP1, and P1 in plasma;

[0042] Figure 8 In Figure A, the fluorescence reproducibility ratios of FRET-ACQ probe, BDP1, and P1 in cells are shown; in Figures B and D, the cell viability of RAW264.7 cells after co-incubation for 24 h with PM labeled with different concentrations of FRET-ACQ probe (B), BDP1 (C), and P1 (D) are shown. mPEG was used as the mPEG index. 2K -PDLLA 2K PM concentration was measured up to 5 mg / mL (FRET-ACQ probes: 20 μM BDP1 and 10 μM P1; BDP1: 20 μM; P1: 10 μM).

[0043] Figure 9In AC, the fluorescence in vivo imaging at different time points after intravenous administration of probe-labeled PM and probe pre-quenching solution to mice (A, FRET-ACQ probe; B, BDP1; C, P1); DF represents the fluorescence intensity (left Y-axis) and reproducibility (right Y-axis) of the easily reproducible liver region in AC.

[0044] Figure 10 AC represents the fluorescence imaging of isolated organs 24 h after intravenous administration of probe-labeled PM and probe pre-quenching solution to mice (A, FRET-ACQ probe; B, BDP1; C, P1); D represents the fluorescence reproduction ratio of the main reproducible organs liver, spleen, lung and kidney.

[0045] Figure 11 In Figure A, fluorescence in vivo imaging of FRET probe-labeled PM and probe pre-quenching solution at different time points after intravenous administration to mice; in Figure B, fluorescence intensity (left Y-axis) and reproduction ratio (right Y-axis) of the easily reproducible liver region in Figure A; in Figure C, in vitro fluorescence intensity of major organs and tissues of FRET probe-labeled PM and probe pre-quenching solution 24 hours after intravenous injection into mice; and in Figure D, fluorescence reproduction ratio of the FRET probe in the major easily reproducible organs of liver, spleen, lung, and kidney. Detailed Implementation

[0046] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0047] To better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the present invention can be understood more clearly and thoroughly, and that the scope of the present invention can be fully conveyed to those skilled in the art.

[0048] Example 1

[0049] This embodiment provides a method for preparing BDP1, the specific steps of which are as follows:

[0050]

[0051] Phosphorus oxychloride (1.5 mmol) was slowly added dropwise to N,N-dimethylformamide (1.5 mmol) in an ice-water bath with stirring. After 10 min, the mixture was stirred at room temperature for 15 min, and then a solution of 7-methoxy-4,5-dihydro-1H-benzo[g]indole-3-phenyl in 1,2-dichloroethane was slowly added dropwise to carry out the Vilsmeier reaction. After the reaction was complete, the organic phase was extracted with ethyl acetate and saturated NaCl aqueous solution, dried over anhydrous Na2SO4 and concentrated. Silica gel column chromatography yielded a white powdery solid, 7-methoxy-4,5-dihydro-1H-benzo[g]indole-3-phenyl-2-carboxaldehyde. Under nitrogen protection, the obtained 7-methoxy-4,5-dihydro-1H-benzo[g]indole-3-phenyl-2-carboxaldehyde (0.45 mmol) and 7-methoxy-4,5-dihydro-1H-benzo[g]indole-3-phenyl (0.45 mmol) were dissolved in redistilled and dried 1,2-dichloroethane. Phosphorus oxychloride (0.54 mmol) was slowly added dropwise. After stirring at room temperature for 12 h, triethylamine (3.4 mmol) and boron trifluoride diethyl ether (3.8 mmol) were added. After stirring at room temperature for 4 h, the reaction solution was concentrated and separated by silica gel column chromatography to obtain product BDP1, a green metallic solid, in 20% yield.

[0052] The proton and carbon NMR spectra of BDP1 are as follows:

[0053] 1 H NMR (600 MHz, CDCl3) δ 8.81 (d, J = 8.9 Hz, 2H), 7.47 – 7.43 (m,4H), 7.39 – 7.34 (m, 6H), 7.07 – 7.03 (m, 1H), 7.01 (dd, J = 8.9, 2.7 Hz,2H), 6.82 (d, J = 2.6 Hz, 2H), 3.90 (s, 6H), 2.89 (t, J = 6.7 Hz, 4H), 2.75(s, 4H). 13 C NMR (151 MHz, CDCl3) δ 160.89, 151.02, 143.07, 138.40, 135.25,133.10, 130.37, 129.77, 129.25, 128.80, 128.04, 121.63, 121.34, 114.42,112.59, 55.48, 31.07, 21.25.

[0054] Example 2

[0055] This embodiment tests the ACQ (aggregation-induced quenching) effect of BDP1 and P1.

[0056] The experimental method was as follows: 200 µL of acetonitrile solution (400 µM) of BDP1 or P1 was added to 4 mL of water-acetonitrile mixed solutions with different water contents. After vortexing for 5 s, the fluorescence emission spectrum was immediately scanned, and the fluorescence intensity at the maximum emission wavelength was measured. The fluorescence intensity in acetonitrile was taken as 100%, and the fluorescence intensity of other water contents was normalized to obtain the fluorescence intensity changes for different water contents.

[0057] like Figure 1 As shown, similar to P1, BDP1 exhibits excellent water quenching characteristics and a significant ACQ effect in the water / acetonitrile system. Its fluorescence intensity is extremely sensitive to water in the environment. When the water content in the system exceeds 45%, the fluorescence intensity decreases rapidly due to aggregation. However, when the water content reaches 65%, the fluorescence is almost completely quenched.

[0058] Example 3

[0059] This embodiment tests the spectral properties of BDP1 and P1.

[0060] (1) Ultraviolet absorption spectroscopy test of BDP1 and P1

[0061] Prepare 0.5 mM solutions of BDP1 and P1 respectively. The molar extinction coefficient can be obtained using the following formula: A = εbc. A represents the absorption intensity, ε is the molar extinction coefficient, c is the concentration of the fluorescent dye, and b is the width of the quartz cell used for detection (usually 1 cm).

[0062] Specific steps: Weigh out dye M×5×10 respectively. -3 mg was transferred to a 10 mL volumetric flask and diluted to volume to obtain a dye stock solution (0.5 mM). 80 μL was taken out and diluted to 4 mL with the same solvent. The solution was then tested using a biconical quartz cell. The maximum absorption wavelength and molar extinction coefficient were finally obtained.

[0063] (2) Fluorescence spectroscopy test of dyes

[0064] The fluorescence quantum yield of BDP1 in chloroform was calculated using the following formula: Yu = Ys·(Fu / Fs)·(As / Au)·(nu) 2 / ns 2Yu, Ys: fluorescence quantum yields of the analyte and reference standard; Fu, Fs: integrated fluorescence intensities of the analyte and reference standard; Au, As: absorbance of the analyte and reference standard at the excitation wavelength (with P4 as the reference); nu, ns: solvent refractive indices of the analyte and reference standard. The absorbance at the excitation wavelength (620 nm) was adjusted to between 0.04 and 0.07 for testing. The fluorescence emission wavelength and fluorescence quantum yield were finally obtained. The fluorescence quantum yield of P1 is referenced (Chem. Eur. J. 2006, 12, 7254-7263).

[0065] The absorption and emission spectra of BDP1 and P1 in chloroform Figure 2 The basic spectral data in chloroform (CHCl3) are shown in Table 1.

[0066] Table 1

[0067]

[0068] From the data in Table 1 and Figure 2 It can be seen that BDP1 has strong absorption in the 600-670 nm range (ε = 157000 M). -1 cm -1 It exhibits bright fluorescence in the 650-750 nm range and a high fluorescence quantum yield (Φ = 0.74). Compared to P1(λ... ab / em Compared to (740 / 755nm), the spectrum of BDP1 shows a significant blue shift, and there is a certain separation between the two spectra. Figure 2 In addition, the emission spectrum of BDP1 and the absorption spectrum of P1 have some overlap, and the two have the potential to form a FRET-ACQ dual-effect probe.

[0069] Example 4

[0070] This embodiment determines the optimal concentrations of BDP1 and P1 in PM labeled with FRET-ACQ dual-effect near-infrared probes (FRET-ACQ probes, including BDP1 and P1) and characterizes PM labeled with the optimal probe concentrations. PM labeled with the probes is prepared using the following method:

[0071] Probe-labeled mPEG was prepared using a thin-film dispersion method. 2k -PDLLA 2k PM, BDP1, P1 and mPEG 2k -PDLLA 2kDissolve in dichloromethane, and remove the solvent by rotary evaporation at 60°C and 85 rpm. Add phosphate buffered saline (PBS) preheated to 60°C, and stir at 60°C (500 rpm) for 30 min. Filter the aqueous solution through a 0.22 μm syringe filter to obtain the probe-labeled PM.

[0072] like Figure 3 As shown in AD, when the concentrations of BDP1 and P1 loaded were in the ranges of 10-40 μM and 5-15 μM, respectively, the FRET-ACQ probe dispersed well in PM, and its absorption was approximately equal to the sum of the absorptions of PM labeled with BDP1 or P1 alone at the corresponding concentrations. The fluorescence re-enhancing interference ratio was the ratio of the fluorescence intensity of the probe after disintegration and redispersal to the fluorescence intensity of the probe-labeled nanocarrier. Considering that the fluorescence re-enhancing signal of the FRET-ACQ probe consisted of the fluorescence re-enhancing signals of BDP1 and P1, increasing the fluorescence intensity of PM labeled with the FRET-ACQ probe compared to BDP1 or P1 reduced the fluorescence re-enhancing interference. Compared to PM labeled with BDP1 alone, the fluorescence of BDP1 decreased significantly after the introduction of the acceptor probe P1, while the fluorescence of P1 appeared, demonstrating the existence of a FRET effect from BDP1 to P1. Figure 4 Although the maximum emission wavelength of the BDP1-labeled PM is 670 nm, due to spectral tailing, it is at 749 nm (λ of P1). em There is still a strong fluorescence signal. Figure 4 P1-labeled PMs, however, suffer from excitation wavelength mismatch (λ). ex =642nm), low excitation efficiency, only a weak signal at 749nm. Furthermore, compared to P1, BDP1-labeled PM has a stronger signal at 749nm, indicating that the fluorescence enhancement of the FRET-ACQ probe relative to BDP1 has a greater impact on reducing fluorescence reproduction interference. Therefore, the fluorescence enhancement factor of the FRET-ACQ probe relative to BDP1 was calculated ( Figure 5 When PM is co-labeled with 20 μM BDP1 and 10 μM P1, PM exhibits moderate fluorescence intensity with a fluorescence intensity enhancement factor of 2.3. Therefore, this concentration is selected as the preferred concentration for labeling PM with the FRET-ACQ probe.

[0073] It should be noted that after BDP1 and P1 are loaded onto PM, the encapsulation efficiency of BDP1 and P1 is close to 100%.

[0074] At room temperature, the above-mentioned preferred concentration labeled PM was diluted with deionized water to 1-5 mg / mL (carrier material concentration), and its particle size and zeta potential were measured. The particle size, PDI, and zeta potential characterization data are listed in Table 2.

[0075] Table 2

[0076]

[0077] The probe labeling has minimal impact on the particle size, PDI, and Zeta potential of PM. After labeling, the PM particle size remains monodisperse within the 16-24 nm range, while the Zeta potential remains within ±3 mV. These results demonstrate that the probes provided by this invention, particularly the FRET-ACQ probe, can be effectively encapsulated within PM for in vivo tracer assays.

[0078] Example 5

[0079] This embodiment tests and compares the fluorescence quenching stability of FRET-ACQ probe, BDP1, and P1 in plasma. The experimental method is as follows: DMSO solutions of the three probes are mixed with mouse plasma at a volume ratio of 1:100 and incubated at 37°C with a shaking speed of 120 rpm. After incubation for different times, samples are taken and the fluorescence intensity is measured. Quenching stability is calculated with the fluorescence intensity of PM labeled with the same concentration of probe as 100%.

[0080] In this embodiment, the DMSO solution of the FRET-ACQ probe has a BDP1 concentration of 200 µM and a P1 concentration of 100 µM, with a BDP1 to P1 ratio of 2:1; the DMSO solution of the BDP1 probe alone has a BDP1 concentration of 200 µM; and the DMSO solution of the P1 probe alone has a P1 concentration of 100 µM.

[0081] like Figure 6 As shown, since BDP1 and P1 can transfer to hydrophobic waters such as plasma proteins, fluorescence reproducibility gradually increases with incubation time. After 24 hours, their fluorescence retention signals are 9% and 15% of those of PM labeled with the same concentration of probe, respectively. This undoubtedly greatly reduces the accuracy of in vivo drug carrier tracing. In contrast, the FRET-ACQ probe provided in this invention has a fluorescence retention rate of only 3% after 24 hours, indicating significantly enhanced quenching stability.

[0082] Example 6

[0083] This embodiment tests and compares the fluorescence reproducibility of the FRET-ACQ probe, BDP1, and P1 water-quenched solutions in plasma. Specifically, DMSO solutions of each probe were vortexed with pure water at a volume ratio of 1:100 to quench fluorescence. The water-quenched solutions were then mixed with mouse plasma at a volume ratio of 1:10 and incubated at 37°C with a shaking speed of 120 rpm. After incubation for different times, samples were taken to measure fluorescence intensity. The percentage of fluorescence reproducibility was calculated with the fluorescence of PM labeled with the probe at the same concentration defined as 100%.

[0084] In this embodiment, the concentrations used for each probe are as follows: After mixing the DMSO solution of the FRET-ACQ probe with 100 times the amount of pure water, the concentration of BDP1 in the water quenching solution is 20 µM, and the concentration of P1 is 10 µM; after mixing the water quenching solution with plasma, the final concentration of BDP1 in the plasma is 2 µM, and the concentration of P1 is 1 µM. Similarly, after mixing the DMSO solution of the BDP1 probe alone with 100 times the amount of pure water, the concentration of BDP1 in the water quenching solution is 20 µM; after mixing the water quenching solution with plasma, the final concentration of BDP1 in the plasma is 2 µM. Likewise, after mixing the DMSO solution of the P1 probe alone with 100 times the amount of pure water, the concentration of P1 in the water quenching solution is 10 µM; after mixing the water quenching solution with plasma, the final concentration of P1 in the plasma is 1 µM.

[0085] like Figure 7 As shown, compared with BDP1 and P1, the water-quenched solution of the FRET-ACQ probe exhibited superior fluorescence reproducibility in plasma. After 24 hours of incubation, the fluorescence reproducibility of BDP1 and P1 was 3% and 5%, respectively; while the fluorescence reproducibility of the FRET-ACQ probe was only 1%.

[0086] Example 7

[0087] This embodiment tests and compares the fluorescence reproducibility of the FRET-ACQ probe, BDP1, and P1 water-quenched solutions in cells. Specifically, Raw264.7 cell suspension (2.5 × 10⁻⁶) was used. 5 Add the probe-labeled PM to a 6-well cell culture plate and incubate for 24 hours to obtain a monolayer of cells. Dilute the probe-labeled PM 100-fold with cell culture medium and co-incubate with the cells. After 4 hours, collect the cells and study the uptake of the probe-labeled PM by Raw264.7 cells using flow cytometry (638nm excitation; FRET-ACQ probe and P1 detection at 750-810nm, BDP1 detection at 700-725nm). Replace the probe-labeled PM with the same concentration of probe water-quenching solution, keeping other operations unchanged, and obtain the corresponding reproducible signal with the water-quenching solution. Calculate the fluorescence reproducibility of the probe in cells by the ratio of the water-quenching solution reproducible signal to the PM uptake signal.

[0088] In this embodiment, the concentration of BDP1 in PM labeled with the FRET-ACQ probe was 20 µM, and the concentration of P1 was 10 µM. After being diluted 100-fold, the final concentration of BDP1 in the PM or water-quenched solution incubated with cells was 0.2 µM, and the concentration of P1 was 0.1 µM. Similarly, the concentration of BDP1 in PM labeled with the BDP1 probe alone was 20 µM, and after being diluted 100-fold, the final concentration of BDP1 in the PM or water-quenched solution incubated with cells was 0.2 µM. Likewise, the concentration of P1 in PM labeled with the P1 probe alone was 10 µM, and after being diluted 100-fold, the final concentration of P1 in the PM or water-quenched solution incubated with cells was 0.1 µM.

[0089] like Figure 8 As shown, the fluorescence reproducibility of the FRET-ACQ probe in Raw264.7 cells was lower (16%) compared to BDP1 (33%) and P1 (45%). Furthermore, Raw264.7 cells exhibited good tolerance to probe-labeled PM, maintaining over 90% cell viability after 24 hours of co-incubation with 10-30 μM probe-labeled PM.

[0090] Example 8

[0091] This embodiment tests the distribution and reproducibility of the FRET-ACQ probe, BDP1 or P1 labeled PM in living tissue prepared in the above embodiments.

[0092] Male C57BL / 6J mice, weighing 20±2g, were selected. Before the experiment, the abdominal hair of the mice was removed to reduce autofluorescence from the hair. All animals were then fasted for 12 hours. After acquiring untreated fluorescence photographs, 0.2 mL of probe-labeled PM (FRET-ACQ group: 20µM BDP1 + 10µM P1; BDP1 group: 20µM BDP1; P1 group: 10µMBDP1) or the same dose of probe pre-quenching solution was injected via the tail vein. Three mice were used in each group. The probe pre-quenching solution was prepared by diluting a high-concentration probe DMSO stock solution 200 times with PBS (pH=7.4) and vortexing to obtain the probe pre-quenching solution. Fluorescence signals at different time points after administration were detected using an IVIS real-time imaging system. Twenty-four hours after administration, the mice were sacrificed, and physiological saline was perfused through the heart to remove blood from the organs and reduce the influence of blood-derived fluorescence. Various organs and tissues were collected, particularly those from the reticuloendothelial system, which are rich in fat and more prone to fluorescence reproducibility. The excitation / emission wavelengths for the FRET-ACQ group were set to 640 / 760 nm, for the BDP1 group to 640 / 680 nm, and for the P1 group to 710 / 760 nm. The average fluorescence intensity in the abdomen of nude mice and in each isolated organ was measured using the system's built-in software. The percentage of fluorescence reproducibility in each tissue, both in vivo and in vitro, was calculated.

[0093] like Figure 9 As shown in AC, the distribution of PM labeled with different probes in vivo was similar. Following intravenous administration, PM fluorescence signals rapidly spread throughout the mouse body and accumulated in the liver until reaching a peak at 8 hours. This is mainly due to the recognition and uptake of PM by the reticuloendothelial system. Subsequently, the PM fluorescence signal in the liver gradually decreased due to clearance. The FRET-ACQ probe pre-quenching solution showed only a weak signal in the easily reproducible liver region within 1 hour. Although this signal gradually increased over time within 8 hours, it remained essentially unchanged thereafter, and the proportion of FRET-ACQ probe fluorescence reproducibility in the liver was less than 15%. Figure 9 (D) indicates that the fluorescence re-enhancing interference of the probe-labeled PM is relatively small. Although the trend of fluorescence re-enhancing signal changes in the liver by the pre-quenching solutions of BDP1 and P1 is similar to that of FRET-ACQ, the proportion of fluorescence re-enhancing of these two probes gradually increases over time, even reaching 32% and 47% respectively after 24 hours of drug administration. Figure 9 EF significantly interfered with real-time in vivo monitoring of PM. Furthermore, in vitro fluorescence imaging 24 hours later showed some recurrence of the pre-quenching solution in the phagocytic organs liver, spleen, lungs, and kidneys. Figure 10AC). Reproducibility studies showed that while the FRET-ACQ probe did not have an advantage in fluorescence reproduction ratios in the spleen, lung, and kidney, its reproduction ratio in the liver, the most important and easily reproduced organ, was significantly lower compared to BDP1 and P1. Figure 10 D). The above results indicate that the FRET-ACQ probe has significant advantages in accurately tracing drug carriers at both in vivo and in vitro levels.

[0094] Comparative Example 1

[0095] This embodiment tests the tissue distribution and fluorescence reproduction of PM labeled with conventional FRET probes (DiD and DiR) in vivo.

[0096] Male C57BL / 6J mice, weighing 20±2g, were selected. Before the experiment, abdominal hair was removed to reduce autofluorescence interference from hair, and all animals were fasted for 12 hours. Background fluorescence images of untreated mice were first acquired. Then, three mice were injected via tail vein with either 0.2 mL of FRET probe-labeled PM (5µM DiD + 5µM DiR) or the same dose of probe pre-quenching solution. After injection, the excitation / emission wavelengths were set to 640 / 780 nm, and the tissue distribution and fluorescence reproduction of the traditional FRET probe were studied using the same imaging procedure as the FRET-ACQ probe in vivo imaging experiment.

[0097] like Figure 11 As shown in A–B, the fluorescence intensity of the traditional FRET probe pre-quenching solution lacking ACQ properties was significantly higher in the liver region than that of the PM group, with its fluorescence reproduction signal exceeding 180% of that of the PM group, indicating extremely severe interference. Further in vitro quantitative results showed that the fluorescence reproduction rate in the liver, spleen, and lungs all exceeded 200%. Figure 11 C–D), which can easily lead to misjudgment of the in vivo fate of nanocarriers. The high fluorescence reproducibility mentioned above mainly stems from the ease with which free and highly dispersed donor and acceptor molecules can re-pair. In contrast, the fluorescence reproducibility of the FRET-ACQ probe is extremely low ( Figure 9 (D and 10D), which may be attributed to the spontaneous aggregation of donor and acceptor after release, limiting their redispersion and thus inhibiting donor-acceptor re-pairing and subsequent fluorescence re-enhancement, demonstrating the significant advantage of FRET-ACQ probes over traditional FRET probes in reducing fluorescence re-enhancement.

[0098] In summary, this invention provides a probe, BDP1, with bright NIR-I region fluorescence and excellent ACQ properties. This probe can be used as a FRET-ACQ dual-effect near-infrared probe, composed of a donor probe and a acceptor ACQ probe, P1, to combine the ACQ principle with another environmentally responsive principle—FRET. When labeled with PM, the FRET-ACQ probe exhibits higher quenching stability and lower fluorescence reproducibility in plasma compared to single ACQ probes, BDP1, or P1. Furthermore, the FRET-ACQ probe shows lower fluorescence reproducibility in cells, in vivo, and in vitro organs, thus offering higher accuracy for in vivo fluorescence tracking of drug carriers.

[0099] Furthermore, the experiment revealed that the near-infrared fluorescent dye provided by this invention did not exhibit significant photoquenching or photobleaching phenomena under normal operating conditions and in an environment without light protection, indicating that its chemical and optical properties are highly stable.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A BODIPY fluorescent dye, characterized in that, It has the structure shown in Equation 1: ； Wherein, R is an alkyl group with 1 to 5 carbons.

2. A method for preparing the BODIPY fluorescent dye as described in claim 1, characterized in that, Includes the following steps: S1. In an ice-water bath, phosphorus oxychloride is added dropwise to an equimolar amount of N,N-dimethylformamide. After stirring for 10-15 minutes, the mixture is brought to room temperature and stirred for another 15-20 minutes to obtain the first reaction solution. S2. Add a 1,2-dichloroethane solution of 7-alkoxy-4,5-dihydro-1H-benzo[g]indole-3-phenyl to the first reaction solution to carry out the Vilsmeier reaction, and synthesize and isolate the product 7-alkoxy-4,5-dihydro-1H-benzo[g]indole-3-phenyl-2-carboxaldehyde. S3. Under an inert gas atmosphere and at room temperature, 7-alkoxy-4,5-dihydro-1H-benzo[g]indole-3-phenyl-2-carboxaldehyde and an equimolar amount of 7-alkoxy-4,5-dihydro-1H-benzo[g]indole-3-phenyl are dissolved in redistilled and dried 1,2-dichloroethane. Phosphorus oxychloride is then added dropwise, and the mixture is stirred for 12-14 hours. Triethylamine and boron trifluoride diethyl ether are then added, and the mixture is stirred at room temperature for 4-5 hours to obtain the second reaction solution. The alkoxy group is an alkoxy group with 1-5 carbon atoms. S4. The second reaction solution was concentrated and subjected to column chromatography to separate a green metallic solid, which was the BODIPY fluorescent dye shown in Formula 1.

3. The application of the BODIPY fluorescent dye as described in claim 1 in the fluorescence tracing of nanocarriers in biological organisms.

4. The application as described in claim 3, characterized in that, Using BODIPY fluorescent dye as the donor ACQ probe and P1 as the acceptor ACQ probe, the BODIPY fluorescent dye and P1 form a FRET-ACQ dual-effect near-infrared probe, which is used for fluorescence tracing of nanocarriers in vivo; the structural formula of P1 is shown in Formula 2: 。 5. The application as described in claim 4, characterized in that, When the FRET-ACQ dual-effect near-infrared probe is loaded on a nanocarrier, the concentration of BODIPY fluorescent dye used is 10-40 μM, and the concentration of P1 used is 5-15 μM.

6. The application as described in claim 4, characterized in that, The FRET-ACQ dual-effect near-infrared probe is encapsulated within a nanocarrier; The nanocarrier has a hydrophobic matrix structure or a hydrophobic core-hydrophilic shell structure; The FRET-ACQ dual-effect near-infrared probe is encapsulated in a hydrophobic matrix or hydrophobic core of a nanocarrier.

7. The application as described in claim 6, characterized in that, The hydrophobic matrix structure is at least one of polymer nanoparticles / microspheres, solid lipid microparticles / nanoparticles, microemulsions, nanoemulsions, and nanocrystals. The hydrophobic core-hydrophilic shell structure is a polymer nanomicelle.

8. The application as described in any one of claims 4-7, characterized in that, The nanocarrier is at least one of the following: polymeric micelles, polymeric nanoparticles, solid lipid nanoparticles, nanoemulsions, inorganic nanoparticles, nanoemulsions, and nanocrystals.

9. The application as described in claim 8, characterized in that, The nanocarrier is a nanoscale material used for drug delivery or gene delivery.

10. A FRET-ACQ dual-effect near-infrared probe, characterized in that, include: The donor ACQ probe and the acceptor ACQ probe are a BODIPY fluorescent dye with the structure shown in Formula 1 and a compound with the structure shown in Formula 2. Equations 1 and 2 are shown below: ， 。

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