Preparation method, nano - complex and application of a squarylium fluorescent imaging reagent in the second near - infrared region

Abstract

The present invention discloses a preparation method, a nano - composite and an application of a squarylium - based fluorescent imaging reagent in the second near - infrared region, belonging to the field of nanomaterials. The squarylium - based fluorescent molecule in the second near - infrared region of the present invention is a class of organic small - molecule compounds with good photo - stability, relatively large molar extinction coefficient, strong fluorescence emission, and can effectively overcome the defect of low fluorescence quantum yield of traditional second - near - infrared - region fluorescent dyes. The present invention uses commercially available bovine serum albumin to encapsulate the fluorescent molecule to form a nano - composite. The prepared fluorescent probe has advantages such as good photo - stability, excellent water - solubility and biocompatibility, deep tissue penetration ability, extremely high signal - to - noise ratio of biological imaging, and extremely high sensitivity. The second - near - infrared - region fluorescent probe provided by the present invention is more promising for future in - vivo imaging, early tumor diagnosis and surgical navigation, can play an important role in future medical optical examinations, and has excellent application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of nanobiomedical imaging, and particularly relates to a preparation method of a squarylium-based fluorescent imaging reagent in the second near-infrared region, a nanocomposite, and their applications. Background Art

[0002] Fluorescence imaging has currently become a basic detection method in biomedicine. Its principle is mainly to obtain the fluorescence signal of biological tissues through the interactions of photons with different tissues of organisms, such as reflection, scattering, absorption, and autofluorescence. Fluorescence imaging has obvious advantages such as fast signal feedback speed, strong multi-signal acquisition ability, high sensitivity, and no ionizing radiation, and can deeply understand the anatomical structure and physiological activity level of living organisms. The wavelengths of excitation and emission in classical fluorescence imaging are mainly in the visible light region, with wavelengths mainly in the range of 400 - 650 nm. However, due to the significant absorption, scattering effects, and strong autofluorescence of biological tissues, the tissue penetration ability of this type of fluorescence imaging is relatively low, and it cannot fully reflect physiological and pathological information. The second near-infrared region (NIR-II) has wavelengths mainly in the range of 1000 - 1700 nm. It has the least light-tissue interaction, shows low photon scattering, and has weak tissue autofluorescence in this region, which greatly improves the detection depth, resolution, and sensitivity of fluorescence imaging.

[0003] In view of the requirements of its biological applications, most of the currently reported NIR-II small molecules still have the following problems that need to be solved urgently: (1) the biosafety of NIR-II fluorescence imaging reagents; (2) their optical properties are still generally low; (3) insufficient modifiability and compatibility. Summary of the Invention

[0004] To solve the above problems, the purpose of the present invention is to solve the problems of insufficient modifiability of existing donor-acceptor-donor (D-A-D) type molecules in the second near-infrared region, single structure of the acceptor unit, and weak fluorescence intensity in the second near-infrared region. A squarylium-based fluorescent imaging reagent in the second near-infrared region is developed, and a nanocomposite in the second near-infrared region is prepared using this reagent, aiming to improve the fluorescence intensity of the fluorescence probe for imaging in the second near-infrared region and achieve a clear and accurate in-vivo second near-infrared region vascular imaging effect.

[0005] To solve the above technical problems of the present invention, the present invention specifically adopts the following technical solutions:

[0006] The present invention provides a squarylium-based fluorescent imaging reagent in the second near-infrared region, and its molecular structural formula is as follows:

[0007] ;

[0008] wherein, R1 is an alkyl group with 1 - 20 carbon atoms; R2 is any one of the following groups:

[0009] 。

[0010] Preferably, the molecular structural formula of the squarylium fluorescent imaging reagent in the second near-infrared region is as follows:

[0011] 。

[0012] The present invention also provides a preparation method of the above-mentioned squarylium fluorescent imaging reagent in the second near-infrared region, which uses squaric acid - malononitrile as an electron acceptor structural unit and couples it with a large-sized electron donor unit to prepare the squarylium fluorescent imaging reagent in the second near-infrared region.

[0013] Preferably, the structural formula of the squaric acid - malononitrile is: ;

[0014] The structural formula of the large-sized electron donor unit is: ;

[0015] Among them, R1 is an alkyl group with 1 - 20 carbon atoms; R2 is any one of the following groups:

[0016] ;

[0017] The specific synthesis steps are as follows: After heating and refluxing the toluene / n-butanol solution of the large-sized electron donor unit and squaric acid - malononitrile, the solvent is removed and purified to obtain the target product; the synthesis route is:

[0018] 。

[0019] Preferably, in the synthesis steps, the molar ratio of the large-sized electron donor unit to squaric acid - malononitrile is 2:1; the volume ratio of the reaction solvent toluene / n-butanol is 1:1; the purification is carried out by silica gel column chromatography, and its mobile phase is analytical pure petroleum ether:ethyl acetate (V / V) = 5:1.

[0020] Preferably, the large-sized electron donor unit is prepared by the following method:

[0021] S1. Take bromo-1,8-naphthalimide, bromoalkane, and potassium carbonate and dissolve them in acetonitrile. After refluxing the mixture for 72 h, the solvent is removed, and it is extracted with ethyl acetate and water. The organic layer is dried and filtered, and the solvent is removed and purified to obtain the compound of formula (2);

[0022] S2, the compound of formula (2), diphenylamine substituted with different R2 groups, sodium tert-butoxide (t-BuONa), tridibenzylideneacetone dipalladium (Pd2(dba)3) and tri-tert-butylphosphine ((t-Bu)3P) are added to a toluene solution in sequence, stirred at 110°C under argon for 12 h, then cooled to room temperature to remove the solvent, extracted with ethyl acetate and water, dried and filtered the organic layer, removed the solvent, and purified to obtain the compound of formula (3);

[0023] S3, adding a tetrahydrofuran solution of methylmagnesium chloride dropwise to an anhydrous tetrahydrofuran solution of the compound of formula (3) under ice-water bath conditions, then heating to 60°C and stirring for 2 h, continuing to cool to 0°C, adding water and perchloric acid solution, extracting with dichloromethane and water, drying and filtering the organic layer, and removing the solvent to obtain a compound of formula (4) which is a large-sized electron donor unit;

[0024] The reaction formula is as follows:

[0025] .

[0026] Preferably, the molar ratio of brominated 1,8-naphtholactimide, bromoalkane and potassium carbonate in S1 is 1:3:5, and the purification is carried out by silica gel column chromatography, and the mobile phase is analytical grade petroleum ether: ethyl acetate (V / V) = 50:1; the molar ratio of the compound of formula (2), diphenylamine, sodium tert-butoxide, tridibenzylideneacetone dipalladium and tri-tert-butylphosphine in S2 is 1:1:3:0.05:3, and the purification is carried out by silica gel column chromatography, and the mobile phase is analytical grade petroleum ether: ethyl acetate (V / V) = 30:1; the concentration of the tetrahydrofuran solution of methyl magnesium chloride in S3 is 1 mol / L, and the molar ratio of methyl magnesium chloride to the compound of formula (3) is 3:1.

[0027] The present invention also provides the use of the near-infrared second-zone squaraine-type fluorescent imaging agent obtained by the above preparation method in the preparation of near-infrared second-zone nanocomposites for vascular imaging.

[0028] Preferably, the method for preparing the near-infrared second-region nanocomposite comprises the following steps:

[0029] Under ultrasonic treatment, an N,N-dimethylformamide solution of a near-infrared second-zone square cyanine type fluorescent imaging reagent (NSQ) was added to an ultrapure aqueous solution containing bovine serum albumin (BSA), and the resulting dispersion was heated under ultrasonic conditions for 10 min. Then, an aqueous glutaraldehyde solution was added and stirred at room temperature for 12 h to obtain an optically clear NSQ@BSA nanocomposite aqueous solution. The aqueous solution was filtered and concentrated to obtain a purified NSQ@BSA nanocomposite.

[0030] Preferably, the volume ratio of the N,N-dimethylformamide solution of NSQ to the ultrapure aqueous solution containing BSA is 1:(4 - 6); the molar ratio of NSQ to BSA is 1:1; the mass fraction of the glutaraldehyde aqueous solution is 25%; ultrafiltration centrifugal tubes with a molecular weight cut-off of 100 kDa are used for concentration.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] The squarylium cyanine type fluorescent molecule in the second near-infrared region provided by the present invention is a class of organic small molecule compounds with good photostability, a relatively large molar extinction coefficient, strong fluorescence emission, and can effectively overcome the defect of low fluorescence quantum yield of traditional second near-infrared fluorescent dyes. Using commercially available bovine serum albumin (BSA) to encapsulate the fluorescent molecule to form a nanocomplex, the prepared fluorescent probe has good photostability, excellent water solubility, biocompatibility, and has advantages such as deep tissue penetration ability, extremely high signal-to-noise ratio of biological imaging, and extremely high sensitivity.

[0033] The present invention provides a squarylium cyanine type photothermal reagent in the second near-infrared region, which has greatly increased modifiability and good biocompatibility. After using commercially available bovine serum albumin to encapsulate the fluorescent molecule to form a nanocomplex, when the second near-infrared fluorescent reagent is photoexcited, since the non-radiative transition rate is greatly inhibited, the excited state energy is still dissipated in the form of fluorescence, so it still has a relatively high fluorescence quantum yield in the near-infrared region. Therefore, it has extremely high signal-to-noise ratio of biological imaging and extremely high sensitivity in subsequent in vivo vascular imaging. Description of the Drawings

[0034] Figure 1 1H NMR spectrum of the squarylium cyanine type photothermal reagent in the second near-infrared region in Example 1 of the present invention.

[0035] Figure 2 13C NMR spectrum of the squarylium cyanine type photothermal reagent in the second near-infrared region in Example 1 of the present invention.

[0036] Figure 3 High-resolution mass spectrum of the squarylium cyanine type photothermal reagent in the second near-infrared region in Example 1 of the present invention.

[0037] Figure 4 UV-Vis-NIR absorption spectrum and fluorescence spectrum of the squarylium cyanine type photothermal reagent in the second near-infrared region in Example 1 of the present invention in toluene.

[0038] Figure 5 UV-Vis-NIR absorption spectrum of the NSQ@BSA nanocomplex in Example 2 of the present invention.

[0039] Figure 6Transmission electron microscope photograph and particle size analysis chart of the NSQ@BSA nanocomposite in Example 2 of the present invention.

[0040] Figure 7 In vivo vascular imaging pictures of mice using the NSQ@BSA nanocomposite in Example 3 of the present invention. Detailed implementation manners

[0041] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field of the present invention. The terms used in the description of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0043] Example 1 Near-infrared second-region squarylium-based fluorescence imaging reagent

[0044] The molecular structure of the near-infrared second-region squarylium-based fluorescence imaging reagent in this example is as follows:

[0045]

[0046] The specific synthesis route is as follows:

[0047]

[0048] The preparation method of this example includes the following steps:

[0049] 1) Dissolve the compound of formula (1) (5 mmol, 1.24 g), 1-bromo-2-octyldodecane (15 mmol, 5.4 g), and potassium carbonate (25 mmol, 3.4 g) in 50 mL of acetonitrile. After refluxing the mixture for 72 h, the solvent is removed by evaporation under reduced pressure, and the mixture is extracted three times with 50 mL each of ethyl acetate and water; the organic layer is dried with sodium sulfate and filtered, and the solvent is removed by evaporation under reduced pressure to obtain a crude product, which is purified by silica gel column chromatography. The mobile phase of the column chromatography is analytical pure petroleum ether:ethyl acetate (V / V) = 50:1, and the compound of formula (2) is obtained as a yellow oily liquid (2.12 g, 80.1%).

[0050] 2) 4,4′-dimethoxydiphenylamine (1 mmol, 229 mg), compound of formula (2) (1 mmol, 529 mg), sodium tert-butoxide (3 mmol, 288 mg), tridibenzylideneacetone dipalladium (0.05 mmol, 45 mg) and tri-tert-butylphosphine (3 mmol, 606 mg) were added to 25 mL toluene solution in sequence. The mixture was stirred at 110°C for 12 h under argon. After the reaction was completed, the mixture was cooled to room temperature and evaporated to remove the solvent under reduced pressure. The obtained mixture was extracted with ethyl acetate and water three times, each time with 50 mL. The organic layer was dried over sodium sulfate and filtered. The solvent was evaporated to remove the crude product under reduced pressure, which was purified by silica gel column chromatography. The mobile phase of the column chromatography was analytical grade petroleum ether: ethyl acetate (V / V) = 30:1 to obtain compound of formula (3) as a red oily liquid (0.45 g, 66.5%).

[0051] 3) A tetrahydrofuran solution of methylmagnesium chloride (1 mol / L, 3 mL) was added dropwise to a tetrahydrofuran solution of the compound of formula (3) (1 mmol, 704 mg) in an anhydrous tetrahydrofuran solution in an ice-water bath. After the addition was complete, the reaction system was heated to 60°C and stirred for 2 h. After the reaction was complete, the reaction system was cooled to 0°C, 0.5 mL of water was added, and then 2 mL of a 70% mass concentration perchloric acid solution was added to the reaction mixture to obtain a dark blue solution; then the solution was extracted with dichloromethane and water three times, 50 mL each time. The organic layer was dried over sodium sulfate and filtered, and the solvent was evaporated under reduced pressure to obtain a crude product, which was a blue-black solid compound of formula (4); the crude product was directly used in the next step without purification.

[0052] 4) A solution of the compound of formula (4) (2 mmol, 1.54 g) and squaryl-malononitrile (1 mmol, 291 mg) in 20 mL toluene / n-butanol (10 mL+10 mL) was heated to reflux for 2 h in a Dean-Stark apparatus. After the reaction was completed, the solvent was evaporated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (analytical grade petroleum ether: ethyl acetate = 5:1) to obtain a black solid near-infrared second-zone squaryl cyanine type fluorescence imaging reagent (1.15 g, 78.2%).

[0053] The obtained near-infrared second-zone aggregation-induced emission molecules were characterized by nuclear magnetic resonance hydrogen / carbon spectrum and high-resolution mass spectrometry. The results are shown in the attached Figure 1 , Attachment Figure 2 and attached Figure 3As shown. The obtained squarylium-based fluorescent imaging reagent in the second near-infrared region was tested for its ultraviolet-visible-near-infrared absorption spectrum, and at the same time, the fluorescence emission spectrum was tested using a 1064 nm laser for excitation. Its emission peak was located at 1060 nm, belonging to the materials in the second near-infrared region, as shown in the appendix Figure 4 as shown.

[0054] Example 2 Preparation of a Near-Infrared Region II Squarylium-Based Photothermal Reagent Fluorescent Probe and BSA to Form a Near-Infrared Region II Nanocomposite

[0055] Dissolve NSQ (2.3 mg) in N,N-dimethylformamide (DMF, 1 mL). Under ultrasonic treatment, add the DMF solution of NSQ to an aqueous solution of ultrapure water (5 mL) containing an equimolar amount of BSA (100 mg). The obtained dispersion was heated under ultrasonic conditions for 10 min, and then an aqueous solution of glutaraldehyde (mass fraction 25%, 10 μL) was added and stirred at room temperature for 12 h; an optically clear aqueous solution of NSQ@BSA nanocomposite was obtained. This aqueous solution was filtered through a filter membrane with a pore size of 220 nm, and after filtration, an ultrafiltration centrifuge tube was used to remove the aqueous solution (6000 r / min, 20 min). The concentrated NSQ@BSA nanocomposite was redissolved in ultrapure water (2 mL) and the above operation was repeated twice to obtain a purified NSQ@BSA nanocomposite.

[0056] The absorption spectrum of the nanocomposite was tested by an ultraviolet-visible-near-infrared absorption spectrometer, and the fluorescence spectrum in the second near-infrared region of the nanocomposite was tested by a steady-state fluorescence spectrometer. The results are as shown in the appendix Figure 5 as shown, indicating that the nanocomposite has strong absorption and fluorescence emission in the second near-infrared region.

[0057] The size and morphology of the obtained near-infrared region II nanocomposite were characterized by transmission electron microscopy and particle size analysis respectively, as Figure 6 shown. The particle size analysis data of the nanoparticles showed that their hydrated particle size was 120 nm, while the TEM images showed that the nanoparticles were spherical with a diameter of 90 nm.

[0058] Application Example Application of NSQ@BSA Nanocomposite in Near-Infrared Region II Fluorescent In Vivo Vascular Imaging of Mice

[0059] Tumor-bearing nude mice were injected with the near-infrared region II nanocomposite (prepared in Example 2) via the tail vein.

[0060] The mice were imaged using a near-infrared region II imager, as shown in the appendix Figure 7As shown, it can be seen that 1 minute after tail vein injection, the nanocomposite distributes in the blood vessels throughout the body of the mouse as the blood flows throughout the body. Due to the penetrability of the fluorescence in the second near-infrared region, it can penetrate the tissues and skin of the mouse, and a bright blood vessel outline can be observed.

[0061] The embodiments described above only represent several preferred embodiments of the present invention. The description is relatively specific and detailed, but it is not used to limit the present invention. It should be noted that for those skilled in the art, the present invention can also have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the concept and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A near-infrared second-zone square acid cyanine type fluorescent imaging reagent, whose molecular structure is as follows: 。 2. The preparation method of the near-infrared second-region squarylium fluorescent imaging reagent according to claim 1, characterized in that, The near-infrared second-zone squaraine-type fluorescent imaging reagent is prepared by using squaraine-malononitrile as an electron acceptor structural unit and carrying out a coupling reaction with a large-size electron donor unit; Among them, the structural formula of the squaric acid - malononitrile is: ; the structural formula of the large - size electron - donor unit is: .

3. The preparation method of the near-infrared second-region squarylium-based fluorescent imaging reagent according to claim 2, wherein, The specific synthesis steps of the near-infrared second-zone squaryl cyanine fluorescent imaging agent are as follows: heating a toluene / n-butanol solution of a large-size electron donor unit and squaryl cyanine-malononitrile under reflux, removing the solvent and purifying to obtain a target product; The synthetic route is: 。 4. The preparation method of the near-infrared region squarylium fluorescent imaging reagent according to claim 3, wherein, The molar ratio of the large-size electron donor unit and the square acid-malononitrile in the synthesis step is 2:1; the volume ratio of toluene / n-butanol in the reaction solvent is 1:1; and silica gel column chromatography is used for purification, and the mobile phase is analytical pure petroleum ether: ethyl acetate = 5:1 in volume ratio.

5. The preparation method of the near-infrared second-region squarylium fluorescent imaging reagent according to claim 3, wherein, The large-size electron donor unit is prepared by the following method: S1, dissolving bromo-1,8-naphtholactimide, 1-bromo-2-octyldodecane and potassium carbonate in acetonitrile, reflux the mixture for 72 h, remove the solvent, extract with ethyl acetate and water, dry and filter the organic layer, remove the solvent, and purify to obtain a compound of formula (2); S2, adding the compound of formula (2), 4,4'-dimethoxydiphenylamine, sodium tert-butoxide, tridibenzylideneacetone dipalladium and tri-tert-butylphosphine to a toluene solution in sequence, stirring and reacting at 110°C under argon for 12 h, then cooling to room temperature to remove the solvent, extracting with ethyl acetate and water, drying and filtering the organic layer, removing the solvent, and purifying to obtain the compound of formula (3); S3, adding a tetrahydrofuran solution of methylmagnesium chloride dropwise to an anhydrous tetrahydrofuran solution of the compound of formula (3) under ice-water bath conditions, then heating to 60°C and stirring for 2 h, continuing to cool to 0°C, adding water and perchloric acid solution, extracting with dichloromethane and water, drying and filtering the organic layer, and removing the solvent to obtain a compound of formula (4) which is a large-sized electron donor unit; The reaction formula is as follows: 。 6. The preparation method of the near-infrared second-region squarylium-based fluorescent imaging reagent according to claim 5, wherein, The molar ratio of brominated 1,8-naphtholactimide, 1-bromo-2-octyldodecane and potassium carbonate in S1 is 1:3:5, and the purification is carried out by silica gel column chromatography, and the volume ratio of the mobile phase is analytical grade petroleum ether: ethyl acetate = 50:1; the molar ratio of the compound of formula (2), 4,4'-dimethoxydiphenylamine, sodium tert-butoxide, tridibenzylideneacetone dipalladium and tri-tert-butylphosphine in S2 is 1:1:3:0.05:3, and the purification is carried out by silica gel column chromatography, and the volume ratio of the mobile phase is analytical grade petroleum ether: ethyl acetate = 30:1; the concentration of the tetrahydrofuran solution of methyl magnesium chloride in S3 is 1 mol / L, and the molar ratio of methyl magnesium chloride to the compound of formula (3) is 3:

1.

7. Use of the near-infrared zone II squaraine type fluorescent imaging agent obtained by the preparation method according to any one of claims 2 to 6 in the preparation of near-infrared zone II nanocomposites for vascular imaging.

8. The application according to claim 7, wherein The preparation method of the near-infrared second zone nanocomposite comprises the following steps: Under ultrasonic treatment, an N,N-dimethylformamide solution of a near-infrared second-zone square cyanine-type fluorescent imaging reagent was added to an ultrapure aqueous solution containing bovine serum albumin. The resulting dispersion was heated under ultrasonic conditions for 10 min, and then an aqueous glutaraldehyde solution was added and stirred at room temperature for 12 h to obtain an optically clear NSQ@BSA nanocomposite aqueous solution. The aqueous solution was filtered and concentrated to obtain a purified NSQ@BSA nanocomposite.

9. The application according to claim 8, wherein The volume ratio of the N,N-dimethylformamide solution of the near-infrared second-zone square cyanine type fluorescence imaging reagent to the ultrapure aqueous solution containing bovine serum albumin is 1:(4-6); the molar ratio of the near-infrared second-zone square cyanine type fluorescence imaging reagent to bovine serum albumin is 1:1; the mass fraction of the glutaraldehyde aqueous solution is 25%; and the concentration is carried out using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa.

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