Aza-BODIPY compound and its application in nanoparticle tracing in vivo

Abstract

The present invention relates to a polymethoxy-substituted Aza-BODIPY compound and the application of the Aza-BODIPY compound as an ACQ probe in fluorescence tracing of nanoparticles in vivo, wherein the Aza-BODIPY probe has the following characteristics: 1) Aza-BODIPY has a methoxy group in the para position of the benzene rings at positions 1, 3, 5, and 7, forming a six-membered ring structure on one side; 2) stable fluorescence is emitted when encapsulated in the hydrophobic core of a nanocarrier; 3) after the nanocarrier is degraded or depolymerized, the probe is released, aggregates when encountering water, and the fluorescence is rapidly and completely quenched; 4) there is no obvious fluorescence recurrence, which can reduce the interference of flat fluorescence recurrence. The "ON→OFF" fluorescence switching mode can effectively eliminate the interference of free probe signals, making real-time monitoring of nanocarrier particles in vivo and in vitro more accurate. The method of the present invention can currently be applied to nanocarriers with a highly hydrophobic matrix (lipid or polyester nanoparticles) or a hydrophobic core-hydrophilic shell structure (micelles).

Description

Technical Field

[0001] The invention relates to the field of fluorescent dyes, in particular to an Aza-BODIPY compound and application thereof in in vivo tracing of nanoparticles. Background Art

[0002] In recent decades, nanomedicine has received extensive attention due to its great potential in delivering drugs to target sites accurately and across barriers. Unlike traditional drugs, nanomedicine relies on specific nanocarriers to achieve its functions. Many countries have organized and implemented large-scale nanobiomedicine development programs, with multiple nanomedicines entering clinical trials every year. Some milestone nanomedicine products have also been spawned, such as long-circulating liposomes of doxorubicin. Paclitaxel albumin nanoparticles Paclitaxel polymerized micelles wait.

[0003] Despite this, we still know very little about the in vivo behavior of nanodrug delivery systems. At present, our understanding of the in vivo behavior of nanodrug delivery systems is more based on the observation of their biological effects. Therefore, R&D personnel are forced to screen and optimize the prescriptions and processes of nanodrugs through pharmacological and efficacy tests, using a trial-and-error approach, which is time-consuming, labor-intensive, and inefficient. If the in vivo behavior of nanodrug carriers can be elucidated (including but not limited to pharmacokinetics, distribution, metabolism, elimination, excretion, etc.), the prescriptions and processes of nanodrugs can be optimized in a targeted manner, avoiding many detours and consuming a large amount of pharmacological and efficacy evaluation resources for inefficient optimization work.

[0004] In order to facilitate the study of the movement and transformation process of nano drug carriers in the body, it is first necessary to accurately trace the nano carriers in the body. Based on safety considerations, the nano drugs that are currently on the market and entering the clinical trial stage are mainly composed of biodegradable organic materials. Since these materials themselves do not have spontaneous signals for in vivo tracing, they are often marked with markers (such as radioactive, fluorescent substances, etc.) for tracing observation. Radioactive labeling is often used for real-time monitoring of encapsulated drugs or materials, while fluorescent labeling is often used to monitor nano carriers.

[0005] Commonly used fluorescent probes can be roughly divided into non-environmentally responsive probes and environmentally responsive probes. Non-environmentally responsive fluorescent probes use common fluorescent probes such as coumarin, DiI and fluorescein (Nature Biotechnology, 2008, 26 (7): 799-807; International Journal of Pharmaceutics, 2012, 426 (1-2): 231-238; Analytical Chemistry, 2017, 89 (10): 5193-5200), etc., and are often labeled by physical encapsulation and covalent binding to study the in vivo process of nanocarriers. However, this method has the disadvantages that the marker is easy to leak, and the free marker signal is difficult to distinguish from the nanocarrier signal. To this end, researchers have further developed probes that respond to the environment.

[0006] Common environmental responsive probes can be divided into three categories: fluorescence resonance energy transfer probes ( The signals of these probes on nanocarriers are different from those when they are free outside, which is helpful to distinguish the signals of free markers from those of nanocarriers. Through the changes of these fluorescent signals, nanocarriers in the body can be tracked more accurately.

[0007] The reported environmentally responsive FRET probes include DiD / DiR or DiO / DiR or Cy5 / Cy5.5. After these probes are co-encapsulated in nanocarriers, the FRET (fluorescence resonance energy transfer) effect occurs, at which time the donor fluorescence is greatly weakened and the acceptor fluorescence is greatly enhanced; when the nanocarrier degrades or disaggregates to release the probe, the distance between the two probe molecules increases (much greater than 10nm), and the FRET effect disappears. At this time, only the donor probe can generate emission light. However, this type of probe has the problem that the fluorescence intensity is not directly related to the concentration of nanocarrier particles, and cannot be used to accurately analyze nanocarrier particles. In addition, most FRET probes are hydrophobic probes, which have strong binding to components such as plasma proteins, which will bring unpredictable false positive results.

[0008] When the environmentally responsive AIE probe is encapsulated in the nanocarrier, it emits light due to "restricted intramolecular motion", while when it is not encapsulated, it is dissolved or diluted by the surrounding environment, causing the fluorescence to disappear or weaken, so it can be used for in vivo tracing of nanocarriers. However, due to their strong hydrophobicity, these probes are also prone to re-aggregation in the body after being released from the carrier, or to binding to plasma proteins or adipose tissue, resulting in fluorescence reappearance. Therefore, the application of AIE probes in monitoring nanocarrier particles in vivo has certain limitations.

[0009] The environmentally responsive ACQ probe method uses the fluorescence "ON→OFF" switching mode to study the in vivo process of nanocarriers, which can avoid the interference of free probes on the carrier particle signal. In addition, a trace amount of probe encapsulation can achieve effective labeling, which has extremely high universality and accuracy in studying the fate of nanocarrier particles. The DiR dye used in previous reports (Biomaterials, 2012, 33(21): 5431-5440; International Journal of Pharmaceutics, 2014, 471(1): 525-535; Journal of Controlled Release, 2016, 238: 58-70; J Biomed Opt, 2016, 21(5): 50901; Macromolecular Bioscience, 2018, 18(1): 1700240; ACS Nano, 2018, 12(7): 6458-6468; Int J Nanomedicine, 2019, 14: 8345-8360; Colloids and Surfaces B: Biointerfaces, 2019, 181: 872-878) has a certain ACQ effect, but there has always been a problem of excessive saturation of the fluorescence signal and large test interference. In addition, the research and development team of this application has been engaged in the research of fluorescent dyes for a long time and accidentally discovered a new ACQ probe P2 based on the Aza-BODIPY core structure (see Figure 2The second chemical structure in the first row) has stable photochemical properties, a wavelength in the near-infrared region, and a high fluorescence quantum yield. P2 is dispersed in the nanocarrier and shows strong fluorescence. When the nanocarrier is destroyed, the P2 molecules enter an environment with a high water content and quickly aggregate, resulting in fluorescence quenching. Based on the method that P2 can be used for real-time imaging of nano drug carriers, the applicant has studied the in vivo processes of different nanocarriers such as liposomes, nanocrystals, lipid nanoparticles, polymer nanoparticles, nanoemulsions and microemulsions, and polymer micelles after oral, intravenous, and transdermal administration. In recent years, dozens of related articles have been published on the method based on the unique ACQ effect of P2 for nanotracing (such as Nanomedicine 2015, 11: 1939-1948; Nanoscale 2016, 8: 7024-7035; J Mater Chem B 2016, 4: 2864-2873, etc.). After in-depth research on P2, the applicant found that the fluorescence quenched P2 would be redistributed by proteins and lipids in the body, resulting in fluorescence reappearance, which to some extent weakened the application advantages of this type of ACQ fluorescent probe. In order to eliminate or reduce this fluorescence reappearance phenomenon, we optimized the chemical structure of the Aza-BODIPY fluorescent probe in order to overcome the shortcomings of the current ACQ-based biological imaging, making it a more accurate tool for monitoring biological samples and nano-drug carriers in the body, further revealing the potential mechanism of nano-drug carriers in the body, and accelerating prescription optimization and conversion to clinical applications. Summary of the invention

[0010] 1. Technical issues to be resolved

[0011] Based on the above situation, the present invention provides an application of a polymethoxy-substituted Aza-BODIPY compound in constructing an ACQ probe and in studying the movement and transformation of nanocarriers in vivo. The Aza-BODIPY compound is an ACQ probe with low fluorescence recurrence, high quantum yield and stable properties, and is suitable for fluorescent tracing or quantitative detection of nanocarriers with a highly hydrophobic matrix (lipid or polyester nanoparticles) or a hydrophobic core-hydrophilic shell structure (micelles) in vivo.

[0012] (II) Technical solution

[0013] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0014] In a first aspect, the present invention provides a polymethoxy-substituted Aza-BODIPY compound having the structural formula shown in Formula 1:

[0015]

[0016] Among them, the Aza-BODIPY compound of the structural formula shown in Formula 1 has a methoxy group at the para position of the 1, 3, 5, and 7 benzene rings, forming a six-membered ring structure on one side.

[0017] Preferably, the synthesis method of the Aza-BODIPY compound of the structural formula shown in Formula 1 is:

[0018] Take 7-methoxy-3-(4-methoxyphenyl)-4,5-dihydro-1H-benzo[g]indole and dissolve it in acetic acid. After sodium nitrite is added at 0°C and the reaction is complete, 2,4-bis(4-methoxyphenyl)-1H-pyrrole is added and acetic anhydride is added dropwise. The reaction is first carried out at room temperature and then heated. The blue solution is neutralized with sodium carbonate solution, extracted with dichloromethane, washed with saturated brine, and the organic phase is dried with anhydrous sodium sulfate. The organic phase is removed by rotary evaporation to obtain a metallic luster product. The product is dissolved in 1,2-dichloroethane, DIPEA and boron trifluoride ether solution are added, the reaction is first carried out at room temperature and then heated. The product is cooled to room temperature, the organic solvent is removed by rotary evaporation, and the product is separated by silica gel column chromatography to obtain a copper-colored solid, which is the Aza-BODIPY compound with the structural formula shown in Formula 1.

[0019] The synthetic route of the Aza-BODIPY compound of the structural formula shown in Formula 1 is as follows:

[0020]

[0021] In a second aspect, the present invention provides the use of an Aza-BODIPY compound having a structural formula shown in Formula 1 as an ACQ probe in the fluorescence tracing of nanoparticles in vivo processes.

[0022] Preferably, the Aza-BODIPY compound of the structural formula shown in Formula 1 is encapsulated in a nanocarrier, and the nanocarrier has a structure of a highly hydrophobic matrix or a hydrophobic core-hydrophilic shell structure; the Aza-BODIPY compound of the structural formula shown in Formula 1 is encapsulated in the highly hydrophobic matrix or hydrophobic core of the nanocarrier.

[0023] Preferably, the highly hydrophobic matrix is ​​lipid or polyester nanoparticles, and the hydrophobic core-hydrophilic shell structure is micelles.

[0024] Preferably, the nanocarrier is at least one of nanopolymer micelles, polymer nanoparticles, solid lipid nanoparticles, nanomicroemulsions, nanoemulsions and nanocrystals.

[0025] (III) Beneficial effects

[0026] The compound of the structural formula shown in Formula 1 is used as a fluorescent indicator. The fluorescent indicator is driven by hydrophobic forces in aqueous solution to form aggregates, and the fluorescence is quenched based on the ACQ effect; when the fluorescent indicator can be distributed into the core of the nanoparticle, it exists in a single molecule excited state, thereby emitting fluorescence, and the fluorescence intensity is positively correlated with the distribution of the nanoparticles in the body. The distribution of the nanoparticles in the body can be tracked by detecting the fluorescence intensity. Compared with traditional fluorescent indicators, the present invention surprisingly found that the Aza-BODIPY compound of the structural formula shown in Formula 1 has low fluorescence reproducibility in the biological environment, greatly reducing the interference of non-nanocarrier signals. In an aqueous solution environment, its fluorescence is completely quenched, and there is no interference with the determination, making it more accurate to track nanoparticles. The Aza-BODIPY compound of the structural formula shown in Formula 1 of the present invention also has better chemical and optical stability than traditional fluorescent indicators. Based on the above characteristics, the compound of the structural formula shown in Formula 1 provided by the present invention has better accuracy, precision, sensitivity and stability than the existing ACQ fluorescent probe, and has a wider range of applications.

[0027] The Aza-BODIPY fluorescent probe of the structural formula shown in Formula 1 of the present invention has a maximum absorption wavelength greater than 700nm and a maximum fluorescence emission wavelength greater than 730nm, which belongs to the near-infrared region. As the measurement wavelength red-shifts, light scattering and background autofluorescence weaken, thereby reducing the interference of light scattering and background autofluorescence on the measurement of nanoparticles' in vivo processes. In addition, the compound has a high quantum yield, which is greater than 0.2 in acetonitrile solution. Its higher quantum yield means that a higher response signal can be obtained using fewer fluorescent probes, which helps to improve the sensitivity of the measurement of nanoparticles' in vivo processes. The Aza-BODIPY fluorescent probe of the structural formula shown in Formula 1 of the present invention has stable chemical properties and very stable optical properties. There is no obvious light quenching and photobleaching in a conventional operating environment, and it does not need to be protected from light during operation. In particular, compared with the existing ACQ probe, the compound of the structural formula shown in Formula 1 has a very low degree of fluorescence reproducibility in a biological environment and is not prone to false positive interference. Traditional fluorescent probes such as ACQ probe P2 (see Figure 2 The second chemical structure in the first row) is more easily adsorbed by living plasma proteins or adipose tissue after coming out of the nanoparticle carrier, resulting in fluorescence reproduction, which leads to inaccurate fluorescence testing and greatly interferes with the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The water quenching spectra of Aza-BODIPY (P2) dye and other 6 common dyes.

[0029] Figure 2The structural formulas of 12 Aza-BODIPY dyes with different structures (including the compound of formula 1 and the compound P2) involved in the water quenching and fluorescence reproduction studies.

[0030] Figure 3 is the normalized fluorescence intensity of Aza-BODIPY fluorescent dye (250 nM) in acetonitrile and aqueous solution.

[0031] Figure 4 The normalized fluorescence intensity graph is shown after 250 nM Aza-BODIPY fluorescent dye was incubated in acetonitrile and surfactant for 0 h and 4 h.

[0032] Figure 5 The normalized absorption fluorescence spectrum of the dye in acetonitrile.

[0033] Figure 6 The fluorescence reproduction of the fluorescently labeled PM in the monolayer cells (IVIS imaging system, compound of formula 1 and P2:λ ex / λ em =710 / 760nm, DiR:λ ex / λ em =745 / 800nm, n=3).

[0034] Figure 7 The fluorescence imaging of PM labeled with dye and water-quenched solution in vitro tissues of fasting rats after intravenous administration; a1-c1) corresponds to the tracer labeled with the compound of formula 1; a2-c2) corresponds to the tracer labeled with P2; a3-c3) corresponds to the tracer labeled with DiR. Average radiation efficiency [(p / s / cm 2 / sr)] / [(Μw / cm 2 )](IVIS imaging system, compound of formula 1 and P2:λ ex / λ em =710 / 760nm, DiR:λ ex / λ em =745 / 800nm, n=3).

[0035] Figure 8 (a) shows the fluorescence in vivo imaging of PM labeled with dye and water quenching solution after intravenous administration in fasting rats; (b) shows the statistical curve of the biodistribution of PM labeled with compound of formula 1 in different organs over time; the average radiation efficiency [(p / s / cm 2 / sr)] / [(Μw / cm 2 )](IVIS Imaging System Structural Formula 1 Compound and P2 Compound: ex / λ em =710 / 760nm, DiR:λ ex / λ em=745 / 800nm, n=3). DETAILED DESCRIPTION

[0036] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings.

[0037] In view of the problem that the existing ACQ fluorescent probe is easily adsorbed by plasma proteins or adipose tissue in vivo outside the nanoparticle carrier and thus has fluorescence reproducibility, the present invention provides a polymethoxy-substituted Aza-BODIPY compound as a new type of ACQ fluorescent probe with low fluorescence reproducibility outside the nanoparticle carrier. Figure 2 The first chemical structure of the first row (the structure shown in Formula 1). The Aza-BODIPY compound has a methoxy group at the para position of the benzene rings at positions 1, 3, 5, and 7, forming a six-membered ring structure on one side.

[0038] In order to fully demonstrate the scope of application of the Aza-BODIPY compound fluorescent indicator, in the following examples, common nanoformulation polymer micelles (PM) are selected as examples for verification and explanation, and six common dye structures such as coumarin, cyanine dye Cy5, Nile red, porphyrin, rhodamine, and 1,3,5,7-tetramethyl BODIPY are selected as comparative examples to test and compare the ACQ properties of each dye to fully demonstrate the advantages of the Aza-BODIPY fluorescent indicator provided by the present invention over other structural dyes.

[0039] In addition, the present invention also selected 9 Aza-BODIPY dyes with similar structures including P2 (ACQ probe P2 based on Aza-BODIPY core structure previously proposed by the research and development team of the present application) as comparative examples, tested and compared the ACQ properties of each Aza-BODIPY dye, so as to fully demonstrate the Aza-BODIPY fluorescent indicator provided by the present invention (see Figure 2 The advantages of the first structure in the first row) over other Aza-BODIPY structures. Nine Aza-BODIPY dyes with similar structures were selected, and the structure, quantity and position were changed by using methoxy-containing aromatic rings as substituents. The effects of non-annular, single-annular and double-annular structures were studied, expanding the scope of investigation.

[0040] In the following experiments, in order to further demonstrate the low fluorescence reproducibility of the Aza-BODIPY dye provided by the present invention at the cell and animal levels, the representative fluorescent dyes P2 and DiR were selected as comparative examples to prepare polymer micelle PM for cell and animal level tests. The following are the details.

[0041] In all the following examples, standard operations and purification methods known to those skilled in the art may be used. Unless otherwise indicated, all temperatures are expressed in degrees Celsius. The structural formula of the compound is 1 H NMR and 13 C NMR was used to determine.

[0042] Example 1

[0043] The preparation route of Aza-BODIPY provided by the present invention is as follows:

[0044]

[0045] The preparation method is:

[0046] Take 7-methoxy-3-(4-methoxyphenyl)-4,5-dihydro-1H-benzo[g]indole (91.5 mg, 0.3 mmol), dissolve it with 2 mL of acetic acid, slowly add sodium nitrite (20.7 mg, 0.3 mmol) at 0°C, after the reaction is complete, add 2,4-bis(4-methoxyphenyl)-1H-pyrrole (83.7 mg, 0.3 mmol), and drop 0.5 mL of acetic anhydride. After reacting at room temperature for half an hour and at 80°C for half an hour, the blue solution is neutralized with sodium carbonate solution, extracted with dichloromethane, washed with saturated brine, and the organic phase is dried over anhydrous sodium sulfate and removed by rotary evaporation to obtain a metallic luster product. The product is dissolved with 1,2-dichloroethane, and DIPEA (0.84 mL, 5.1 mmol) and boron trifluoride ether solution (0.7 mL, 5.7 mmol) are added. The reaction is carried out at room temperature for 30 min and at 80°C for 30 min. The product was cooled to room temperature, the organic solvent was removed by rotary evaporation, and the product was separated by silica gel column chromatography to obtain a copper-red solid with a yield of 46%.

[0047] Product 1 H NMR and 13 The results of C NMR analysis are as follows:

[0048] 1 H NMR (600MHz, CDCl3) δ = 8.72 (d, J = 9.0Hz, 1H), 8.09 (d, J = 8.6Hz, 4H), 7.73 (d, J = 8.4Hz, 2H), 7.06 (dd, J = 8.2, 5.7 Hz,4H),6.97(dd,J=17.4,5.4Hz,3H),6.89(s,1H),6.85(d,J=1.9Hz,1H),4.11–3.81(m,12H),2.96(s,4H)ppm;

[0049] 13C NMR(150MHz, CDCl3)δ=162.69,161.16,155.18,154.67,146.57,144.95, 144.38,140.30,138.39,132.92,132.32,132.11,131.89,131.11,130.4 0,128.87,128.53,128.50,128.40,128.13,124.93,119.96,117.03,114 .50,114.07,113.22,55.47,55.35,30.49,21.80ppm; HRMS(MALDI):[M+H + ]m / z(%):calcd for C 38 H 32 BF2N3O4:644.2527; found:644.2535.

[0050] After analysis, the product has Figure 2 The structural formula shown is the first one in the first row.

[0051] Example 2

[0052] This example tests and compares the ACQ properties of Aza-BODIPY (P2) dye and other 6 common dyes. The experimental method is: take 20 μL, 50 μM of the existing 7 dye samples and add them to 4 mL of water-acetonitrile mixed solution with water content of 0%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 80%, 90%, 95% and 100%, vortex for 5 seconds, immediately measure the fluorescence and scan the fluorescence emission spectrum. The excitation wavelength adopts the maximum absorption wavelength of the dye blue shifted 20nm to obtain a complete fluorescence emission spectrum.

[0053] like Figure 1As shown in the figure, the fluorescence intensity at the maximum emission wavelength of the fluorescent dye changes with the increase of water content. The maximum fluorescence wavelength of coumarin dyes and 1,3,5,7-tetramethyl BODIPY dyes is short, and this type of dye is not suitable for in vivo imaging research. Similar to coumarin dyes, the maximum fluorescence wavelength of Nile Red dyes will show obvious red shift phenomenon depending on the system, but there is overlap with the original spectrum, which will interfere with subsequent imaging applications; Rhodamine B dyes are water-soluble dyes, so their fluorescence shows fluorescence enhancement in solution systems with high water content; the fluorescence of Cy5 dyes and 1,3,5,7-tetramethyl BODIPY dyes cannot be completely quenched, which shows that the ACQ properties of these two dyes are not obvious. The fluorescence emission wavelength of porphyrin dyes is relatively special, showing multiple fluorescence peaks. Phthalocyanine dyes are too hydrophobic to be dissolved in acetonitrile solution, which is not convenient for testing. Aza-BODIPY (P2) dyes have a fluorescence wavelength that does not change with changes in the environmental system, have a strong fluorescence intensity, and have a maximum fluorescence wavelength in the near-infrared region, which is beneficial for biological imaging. In addition, the dye has a fluorescence mutation as the water content of the solution increases, and has good ACQ properties. Therefore, compared with the other six common dyes, Aza-BODIPY (P2) dyes have better ACQ properties.

[0054] Example 3

[0055] This example tests and compares 12 dyes including the Aza-BODIPY compound of formula 1 and the Aza-BODIPY (P2) compound (eg Figure 2 The water quenching properties of the structure shown in FIG. 1 are as follows:

[0056] First, take 20 μL, 50 μM of each dye sample and add it to 4 mL of water and 4 mL of acetonitrile solution respectively. After vortexing for 5 seconds, immediately measure the fluorescence and scan the fluorescence emission spectrum. The excitation wavelength uses the maximum absorption wavelength of the dye to blue-shift by 20 nm to obtain a complete fluorescence emission spectrum. Then, each dye is dissolved in acetonitrile, diluted with pure acetonitrile and pure water respectively, irradiated with the excitation wavelength corresponding to each dye, and the fluorescence intensity of 250 nM dye is tested to determine the water quenching of the dye. Figure 3 As shown, compared with the signal in acetonitrile, the residual fluorescence of compound 5 and compound 7 in pure water is more significant, and the remaining dyes can be completely quenched.

[0057] Example 4

[0058] This example tests and compares 12 dyes including the Aza-BODIPY compound of formula 1 and the Aza-BODIPY (P2) compound (eg Figure 2 The reproducibility of quenching fluorescence of the quenched ...

[0059] Take 0.4mL of 50μM dye acetonitrile solution and vortex mix with 1.6mL of pure water to quench the fluorescence, take 1mL of the quenching solution and mix with 9mL of 1% surfactant (Triton×100 and Tween 80) solution, place in a 37℃ water bath shaker, and take 4mL at 0h and 4h to measure the fluorescence intensity. At the same time, take 1mL of the quenching solution and mix with 9mL of acetonitrile, and measure the fluorescence intensity as a reference. Notes: 1) Some dyes have poor solubility in acetonitrile. After being dispersed after ultrasound, fine granular precipitates will appear after standing overnight, and they need to be completely redissolved before testing; 2) After the dye and surfactant are vortexed, they are placed at room temperature for 30 minutes. After the bubbles disappear and the mixture is stable, measure again to avoid errors. From the measurement results Figure 4 As shown (in each group, the bars from left to right correspond to acetonitrile, Triton×100 0h, Triton×100 4h, Tween 80 0h, Tween 80 4h), among which the fluorescence reappearance of the compound of formula 1 is the weakest.

[0060] Example 5

[0061] In this example, the optical parameters of the compound dye of formula 1 are tested with the P2 compound dye and the DiR dye as references. The experiment includes:

[0062] (1) UV absorption spectrum test of dyes

[0063] Prepare a 0.5 mM solution of the dye to be tested. The molar extinction coefficient can be calculated using the following formula: A = εbc. A represents the absorption intensity, ε is the molar extinction coefficient, c is the concentration of the compound, and b is the width of the quartz cell used for detection (usually 1 cm). -3 mg, transfer to a 10mL volumetric flask, dilute to volume as sample A (0.5mM), take out 80μL → 4mL, and test with a double-sided transparent cuvette. Finally, the maximum absorption wavelength and molar extinction coefficient are obtained.

[0064] (2) Fluorescence spectrum test of dyes

[0065] The fluorescence quantum yield is obtained by the following calculation formula: Yu = Ys Fu / Fs As / Au. Yu, Ys: fluorescence quantum yield of the test substance and the reference standard substance; Fu, Fs: integrated fluorescence intensity of the test substance and the reference substance; Au, As: absorbance of the incident light of the test substance and the reference substance at the excitation wavelength (with P2 dye as reference). The absorbance at the excitation wavelength (380nm) is adjusted to about 0.04-0.07 below 0.1 and tested. Finally, the fluorescence emission wavelength and fluorescence quantum yield are obtained.

[0066] The spectra of the Aza-BODIPY compounds tested are Figure 5 The data of maximum absorption wavelength, fluorescence emission wavelength, Stokes shift, half peak width, molar extinction coefficient and fluorescence quantum yield in acetonitrile are shown in Table 1.

[0067] Table 1 Basic spectral data of dyes in acetonitrile

[0068]

[0069] As can be seen from the above table, the fluorescence quantum yield of the compound of formula 1 is 0.33, which is higher than that of DiR dye. The Aza-BODIPY type fluorescent probe of the structural formula shown in formula 1 has a maximum absorption wavelength greater than 700nm and a maximum fluorescence emission wavelength greater than 730nm, which belongs to the near-infrared region. As the measurement wavelength red-shifts, light scattering and background autofluorescence weaken, thereby reducing the interference of light scattering and background autofluorescence on the measurement of nanoparticles in vivo processes. In addition, the compound has a high quantum yield, which is greater than 0.2 in acetonitrile solution. A higher quantum yield means that a higher response signal can be obtained using fewer fluorescent probes, which helps to improve the sensitivity of the measurement of nanoparticles in vivo processes.

[0070] Example 6

[0071] Nanoparticle tracers were prepared by encapsulating P2 compound dye, DiR dye, and compound dye of formula 1 using nanocarrier PM, and characterization tests were performed on the nanoparticle tracers. The experimental method is as follows:

[0072] (1) Preparation of Nanotracers

[0073] Dye-loaded mPEG was prepared by thin film dispersion method 2k -PDLLA.5k polymer micelles (PM), 100mgmPEG 2k -PDLLA 2.5k110.3 μg of the compound of formula 1 was dissolved in 10 mL of dichloromethane and evaporated under vacuum at 60°C to form a uniform film. The film was hydrated in 20 mL of deionized water at 60°C with gentle stirring (500 rpm) for 1 h. The dispersion was filtered through a 0.22 μm filter to obtain a PM suspension.

[0074] The formulations for other fluorescent probe encapsulation were prepared according to the same procedure, but the molar concentrations of different probes were ensured to be the same. The water-quenched dye solutions of different probes were prepared by diluting the probe stock solutions with ultrapure water to the same molar concentrations. All preparations were stored at 0 °C until use.

[0075] (2) Nanoparticle tracer characterization test

[0076] The particle size and zeta potential of the nanotracers were measured at room temperature. All measurements were performed after diluting the formulations to appropriate concentrations with deionized water. The encapsulation efficiency was determined by ultrafiltration.

[0077] After the fluorescent preparation is diluted, place it in an ultrafiltration tube ( Ultra-0.5, 100KDa) centrifuged (6000rpm) for 20min to obtain a filtrate. Extract and measure the unencapsulated probe in the filtrate, add ether (2mL), fully vortex and stir for 20min, and wait for stratification. Extract 1mL of ether layer, blow dry with nitrogen at 40°C, and then dissolve in 0.5mL of dimethyl sulfoxide to measure the fluorescence intensity. Use the fluorescence intensity standard curve to determine the content of free probe.

[0078] The encapsulation efficiency (EE%) of the fluorescent preparation can be calculated by the formula:

[0079]

[0080] In an aqueous environment, the particle size, polydispersity index, zeta potential, and encapsulation efficiency of different nanocarriers were measured, and the characterization data are listed in Table 2.

[0081] Table 2. PM particle size, polydispersity index and zeta potential

[0082]

[0083] The experimental results in the table above show that the particle size of PM is about 60nm, and the Zeta potential is within the range of ±10mV. PM has a high EE (%) (>95%), and can effectively encapsulate P2 compound, DiR, and Formula 1 compound probes into nanocarrier PM for tracer experimental testing.

[0084] Example 7

[0085] This example tests the fluorescence reproduction of the nanoparticle tracer prepared by the above-mentioned examples, which is prepared by encapsulating the P2 compound, DiR, and the dye of the compound of formula 1, at the cellular level. The experimental method is as follows:

[0086] (1) Preparation of cell materials

[0087] Cell culture process: HeLa (cervical cancer cells), Raw 264.7 (mouse mononuclear macrophages) and Caco-2 (human cloned colon adenocarcinoma cells) cells were used. HeLa and Raw264.7 cells were inoculated in 10 cm culture dishes and cultured in DMEM high glucose medium containing a mixture of 10% fetal bovine serum and 1% penicillin-streptomycin. The incubator was set at 37°C and 5% CO2. The medium was replaced with fresh culture medium once a day. Caco-2 cells require 10% of essential amino acids to be supplemented in the culture medium.

[0088] Cell passaging process: When Raw264.7 cells cover the bottom of the culture dish, discard the culture medium in the dish, add 2mL of PBS buffer, remove the PBS buffer after rinsing, add 2mL of trypsin, put it in the incubator for digestion for 4min, wait until the cells fall off the culture dish under the microscope, add 3mL of culture medium, stop digestion, and transfer the solution in the culture dish to a centrifuge tube. Set the centrifuge to 1000 rpm and centrifuge for 3min. Remove the supernatant, add 4mL of fresh culture medium, gently blow 10 times with a 1mL pipette, and after the cells are dispersed, take 1mL and add it to a new 10cm culture dish, and add 7mL of culture medium. HeLa and Raw 264.7 cells are passaged every 2 days; Caco-2 cells are passaged every 4 days, and the medium is changed once a day.

[0089] (2) Nanotracer cell-level fluorescence reproduction test

[0090] Three cell models, Caco-2, HeLa and Raw 264.7, were established to evaluate the cellular uptake and fluorescence reproduction of different cell types. Caco-2 and HeLa cells were grown at 3.2×10 4 / well in a 96-well plate with black walls and transparent bottom. Raw264.7 cells were seeded at a density of 6.4×10 4 / well was inoculated into a black-walled, transparent-bottomed 96-well plate. After 3 days of culture for HeLa cells and Raw 264.7 cells and 4 days of culture for Caco-2 cells, the medium was changed once a day to form a homogeneous cell monolayer. The cell monolayer was washed three times with Hank's salt solution, and the three fluorescent tracers were diluted 3 times with PBS buffer. 100 μL of the diluted sample was added to the cells and incubated for 2 hours. The dispersion was then discarded and the cell monolayer was washed 3 times with PBS buffer. Total fluorescence was measured using an IVIS real-time imaging system.

[0091] like Figure 6 As shown, in the three cell models, the water-quenched compound of Formula 1 showed the lowest fluorescence recurrence in all cell types (about 2-3%); P2 dispersion (less than 8% in Caco-2, about 3% in HeLa and Raw264.7); DiR showed more significant fluorescence recurrence in all cell models, with about 25.7%, 40.4% and 21.7% recurrence in Caco-2, HeLa and Raw264.7 cell monolayers, respectively.

[0092] Example 8

[0093] This example tests the distribution and reproduction of nanoparticle tracers prepared in the above examples, which are prepared by encapsulating the P2 compound, DiR, and the dye compound of formula 1, in living tissues.

[0094] Male SD rats weighing 180±20g were selected. Before the experiment, the abdominal hair of the rats needed to be removed to reduce the autofluorescence from the hair. After that, all animals were fasted for 12h. After collecting fluorescence photos of untreated rats, the fluorescent tracer and its water-quenched dye solution were injected through the tail vein. The injection amount was calculated according to 0.04285μmol per kilogram of mouse, and three rats were selected for each group. The IVIS real-time imaging system was used to detect the fluorescence signal at 5min, 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 36h, and 48h after administration. The excitation / emission wavelength of the Aza-BODIPY probe was set to 710 / 760nm, and the excitation / emission wavelength of the DiR probe was set to 745 / 800nm.

[0095] Following the same experimental steps, the fluorescent tracer and water quenching solution were injected into the tail vein of SD rats. At 1h, 4h, 12h, and 24h after administration, the rats were killed and perfused with normal saline through the heart to remove the blood in the organs and reduce the influence of blood-borne fluorescence. Various organs and tissues were collected, especially the reticuloendothelial system organs that are rich in fat and more prone to fluorescence reappearance. The fluorescence intensity of different organs and tissues was quantitatively determined by measuring the average radiant efficiency (ARE).

[0096] Figure 7As shown, after intravenous administration, the PM fluorescence signals of the compound of formula 1 (see Figures a1-c1) and P2 markers (see Figures a2-c2) quickly spread throughout the rat. It reached a peak value at about 0.5h in the limb tissue and at 8h in the liver, which was mainly due to the recognition and uptake of PM by the reticuloendothelial system (RES) organs. The fluorescence signal of peripheral tissues in the DiR-labeled (see Figures a3-c3) group reached a maximum value 5min after administration, and the fluorescence signal continued to decline within 48h, while the fluorescence intensity of the liver fluctuated within 2h and reached a peak at 8h. P2 and DiR-labeled nanocarriers advanced and amplified the accumulation of PM signals, which had a certain impact on the study of the fate of nanocarriers. In addition, the P2 and DiR water-quenched groups had obvious fluorescence recurrence, which also interfered with normal testing.

[0097] As shown in (a) and (b) of Figure 8, after 1 hour, PM mainly accumulated in RES organs, including liver, spleen and lung, as well as adipose tissue, heart and kidney. In contrast, the fluorescence intensity of muscle and skin was the lowest; within 24 hours, no PM accumulation was observed in the brain. Fluorescence quantification showed that when labeled with the compound of formula 1, the biodistribution pattern of PM was similar, and the order of accumulation in the tested organs / tissues was liver>lung>spleen>heart≈adipose tissue>kidney. The fluorescence first reached a peak at 4 hours after injection and gradually decreased at subsequent time points, indicating that PM gradually accumulated 4 hours after injection, and then the polymer carrier degraded and the released ACQ probe fluorescence was quenched. It is worth noting that the recurrence after quenching with the compound of formula 1 was the weakest. Although P2 showed the same distribution, fluorescence recurrence occurred in the liver and spleen of the three rats 4 hours after injection; the situation of the DiR probe was even worse, and the fluorescence image of the water quenching group showed that the fluorescence intensity of the liver and spleen continued to increase within 24 hours.

[0098] In summary, the compound probe of formula 1 has enhanced hydrophobicity, a larger conjugated plane structure, and a fluorescence emission wavelength in the near-infrared region. Compared with other molecules, it has great advantages in anti-fluorescence reproduction, which is conducive to more accurate biological imaging.

[0099] In addition, the experiment found that the Aza-BODIPY fluorescent probe with the structure shown in Formula 1 had no obvious photoquenching and photobleaching phenomenon under normal operation and in an environment without light protection, indicating that its chemical and optical properties are very stable.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 polymethoxy-substituted Aza-BODIPY compound, characterized in that: It has the structural formula shown in Formula 1: Formula 1.

2. The method for synthesizing the Aza-BODIPY compound of the structural formula 1 according to claim 1, characterized in that: The synthesis method is: Take 7-methoxy-3-(4-methoxyphenyl)-4,5-dihydro-1 H -Benzo[g]indole was dissolved in acetic acid, sodium nitrite was added at 0°C, and after the reaction was complete, 2,4-bis(4-methoxyphenyl)-1 H -pyrrole, and acetic anhydride is added dropwise, reacted at room temperature first and then heated to react, the blue solution is neutralized with sodium carbonate solution, extracted with dichloromethane, washed with saturated brine, the organic phase is dried with anhydrous sodium sulfate, and the organic phase is removed by rotary evaporation to obtain a metallic luster product, the product is dissolved with 1,2-dichloroethane, DIPEA and boron trifluoride ether solution are added, reacted at room temperature first and then heated to react, the product is cooled to room temperature, the organic solvent is removed by rotary evaporation, and the product is separated by silica gel column chromatography to obtain a red copper solid, which is the Aza-BODIPY compound of the structural formula shown in Formula 1.

3. Use of the Aza-BODIPY compound of formula 1 according to claim 1 as an ACQ probe in the preparation of a fluorescent tracer for nanoparticle in vivo processes.

4. The use according to claim 3, characterized in that: The Aza-BODIPY compound of the structural formula shown in Formula 1 is encapsulated in a nanocarrier, and the nanocarrier has a structure of a highly hydrophobic matrix or a hydrophobic core-hydrophilic shell structure; the Aza-BODIPY compound of the structural formula shown in Formula 1 is encapsulated in the highly hydrophobic matrix or hydrophobic core of the nanocarrier.

5. The use according to claim 4, characterized in that: The highly hydrophobic matrix is ​​lipid or polyester nanoparticles, and the hydrophobic core-hydrophilic shell structure is micelles.

6. The use according to claim 4, characterized in that: The nanocarrier is at least one of nano polymer micelles, polymer nanoparticles, solid lipid nanoparticles, nano microemulsions, nano emulsions and nano crystals.

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