Amphiphilic aza-BODIPY fluorescent dye as well as preparation method and application thereof

Abstract

The invention discloses an amphipathic aza-BODIPY fluorescent dye as well as a preparation method and application of the amphipathic aza-BODIPY fluorescent dye. Two tetraethylene glycol are introduced into an aza-BODIPY fluorescent structure to serve as a hydrophilic structure, four C8 or C18 alkyl chains are introduced to serve as hydrophobic elements, nanoparticles, nanosheets and branched nanorod structures can be assembled in a slow water adding or flash precipitation mode, and the nanostructures have very strong absorption at 808 nm, so that the fluorescence intensity of the aza-BODIPY fluorescent material is greatly improved, and the fluorescence intensity of the aza-BODIPY fluorescent material is greatly improved. The material has an excellent photothermal conversion effect and can be used for photothermal therapy of tumors.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent dyes, and in particular relates to an amphiphilic aza-fluoro-boron fluorescent dye and a preparation method and application thereof. Background Art

[0002] In addition to surgical treatment, chemotherapy, radiotherapy, immunotherapy, photodynamic therapy, gene therapy, etc. have been widely used in the treatment of malignant diseases. Among them, photothermal therapy has attracted widespread attention due to its advantages such as non-invasiveness, low side effects and short treatment time. In particular, organic photothermal agents with strong near-infrared absorption are favored due to their excellent biosafety and deep tissue penetration depth.

[0003] In order to obtain good photothermal therapy effects, photothermal agents need to have high photothermal conversion efficiency, long blood circulation time and excellent chemical and photostability. Traditional organic small molecule photothermal agents usually have a strong hydrophobic structure and need to be loaded in an amphiphilic polymer carrier. During the blood circulation process, amphiphilic polymers are prone to depolymerization, resulting in leakage of loaded dye molecules. And the morphology of nanoparticles will also affect their blood circulation time (RSCAdv., 2018, 8, 8089–8100). Therefore, it is of great significance to design amphiphilic dyes with precise structures that can self-assemble in water to form photothermal agents with specific structures.

[0004] Aza-BODIPY has excellent chemical stability and photostability, and a high molar extinction coefficient. Through reasonable molecular structure design, the absorption and emission wavelengths can be adjusted from near-infrared region 1 to near-infrared region 2. It has been widely used in fluorescence imaging, photodynamic therapy, and photothermal therapy (Coord. Chem. Rev., 2023, 476: 214908; Chem. Soc. Rev., 2020, 49(21): 7533-7567).

[0005] Nevertheless, the widely used aza-fluoroboron fluorescent dyes are highly hydrophobic and form nanoparticles after being loaded with amphiphilic liposomes, making it difficult to control the morphology. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides an amphiphilic nitrogen-fluorine-boron fluorescent dye and a preparation method and application thereof. By introducing two tetraethylene glycols as hydrophilic structures and four C8 or C18 alkyl chains as hydrophobic moieties into the nitrogen-fluorine-boron fluorescent structure, nanoparticles, nanosheets and branched nanorod structures can be assembled by slow addition of water or flash precipitation. These nanostructures have very strong absorption at 808nm and excellent photothermal conversion effect, and can be used for photothermal treatment of tumors.

[0007] The technical solution adopted by the present invention is as follows:

[0008] An amphiphilic aza-fluoro-boron fluorescent dye, the structural formula of the amphiphilic aza-fluoro-boron fluorescent dye is as follows:

[0009]

[0010] Wherein, R is R1 or R2;

[0011] The structural formula of R1 is: This amphiphilic aza-fluoro-boron fluorescent dye is named C8-NBDP-OEG4;

[0012] The structural formula of R2 is: This amphiphilic aza-fluoro-boron fluorescent dye is named C 16 -NBDP-OEG4.

[0013] The C8-NBDP-OEG4 can be assembled into nanoparticles in water.

[0014] The C 18 -NBDP-OEG4 can assemble into nanosheets or branched nanorods in water.

[0015] The present invention also provides a method for preparing the amphiphilic aza-fluoro-boron fluorescent dye, which comprises the following steps:

[0016] 1) subjecting p-aminobenzaldehyde to a halogenation reaction with an alkyl halide to obtain an intermediate I;

[0017] 2) subjecting p-hydroxyacetophenone to a nucleophilic substitution reaction with tetraethylene glycol mono-p-toluenesulfonate to obtain intermediate II;

[0018] 3) reacting intermediate I and intermediate II under the catalysis of a base to obtain intermediate III;

[0019] 4) Intermediate III reacts with nitromethane under the catalysis of a base to obtain intermediate IV;

[0020] 5) Intermediate IV is subjected to coupling reaction with acetamide, and the obtained product is then reacted with boron trifluoride ether to obtain an amphiphilic aza-fluoro-boron fluorescent dye;

[0021] The structural formula of the intermediate I is:

[0022] The structural formula of the intermediate II is:

[0023] The structural formula of the intermediate III is:

[0024] The structural formula of the intermediate IV is:

[0025] Furthermore, the preparation method comprises the following steps:

[0026] 1) dissolving p-aminobenzaldehyde and alkyl halide in DMF, adding DIPEA thereto, reacting at 105-115° C. for 10-12 h under nitrogen atmosphere, and purifying by column chromatography to obtain intermediate I;

[0027] 2) dissolving p-hydroxyacetophenone, tetraethylene glycol mono-p-toluenesulfonate and N,N-diisopropylethylamine in anhydrous DMF, reacting at 105-115° C. for 5-6 hours under a nitrogen atmosphere, and purifying by column chromatography to obtain intermediate II;

[0028] 3) Dissolve intermediate I, intermediate II and sodium hydroxide in ethanol, stir at room temperature for 10 to 12 hours, and purify by column chromatography to obtain intermediate III;

[0029] 4) Intermediate III, nitromethane and sodium hydroxide are dissolved in ethanol, reacted at 65-75° C. for 5-6 hours, and purified by column chromatography to obtain intermediate IV;

[0030] 5) Intermediate IV and acetamide are dissolved in n-butanol, and the reaction is carried out at 105-115° C. for 10-12 h in a nitrogen atmosphere. The product is dissolved in anhydrous dichloromethane, and N,N-diisopropylethylamine is slowly added thereto at -15--25° C. in a nitrogen atmosphere. After stirring evenly, boron trifluoride etherate is slowly added thereto, and the reaction is stirred at room temperature for 10-12 h. The product is purified by column chromatography to obtain an amphiphilic aza-fluoroboron fluorescent dye.

[0031] In step 1), the molar ratio of p-aminobenzaldehyde, alkyl halide and DIPEA is 1:2-5:2-5; and the concentration of p-aminobenzaldehyde in DMF is 0.5-1.5M.

[0032] In step 2), the molar ratio of p-hydroxyacetophenone, tetraethylene glycol mono-p-toluenesulfonate, and N,N-diisopropylethylamine is 1:1-1.5:2-2.5; and the concentration of p-hydroxyacetophenone in anhydrous DMF is 0.1-1.0M.

[0033] In step 3), the molar ratio of intermediate I, intermediate II and sodium hydroxide is 1:1-1.2:2-4; the concentration of intermediate I in ethanol is 0.1-0.5M.

[0034] In step 4), the molar ratio of intermediate III, nitromethane and sodium hydroxide is 1:2-10:0.5-2.0; the concentration of intermediate III in ethanol is 0.1-0.5M;

[0035] In step 5), the molar ratio of intermediate IV, acetic acid amine, N,N-diisopropylethylamine and boron trifluoride ether is 1:10-20:2-4:5-5.5; and the concentration of intermediate IV in n-butanol is 0.01-0.1M.

[0036] The present invention also provides a method for preparing the amphiphilic nitrogen-fluorine-boron fluorescent dye self-assembled nanoparticles of the present invention, wherein C8-NBDP-OEG4 is dissolved in dimethyl sulfoxide, which is quickly added into deionized water with high-speed stirring, and then the organic solvent is removed by dialysis to obtain uniform nanoparticles.

[0037] The present invention also provides a method for preparing the amphiphilic nitrogen-fluorine-boron fluorescent dye self-assembled nanosheets of the present invention, wherein C 18 -NBDP-OEG4 is dissolved in N,N-dimethylformamide, deionized water is slowly added thereto, and then the organic solvent is dialyzed to obtain uniform nanosheets.

[0038] Furthermore, the addition rate of the deionized water is 1.5 to 2.5 mL / h.

[0039] The present invention also provides a method for preparing the amphiphilic aza-fluoro-boron fluorescent dye self-assembled branched nanorods of the present invention, wherein C 18 -NBDP-OEG4 was dissolved in dimethyl sulfoxide, deionized water was slowly added thereto, and then the organic solvent was removed by dialysis to obtain branched nanorods.

[0040] Furthermore, the addition rate of the deionized water is 0.5 to 1.5 mL / h.

[0041] The present invention also provides the use of the amphiphilic aza-fluoro-boron fluorescent dye as a photothermal agent.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The amphiphilic aza-fluoro-boron fluorescent dye provided by the present invention can be assembled into nanoparticles, nanosheets and branched nanorod structures by introducing two tetraethylene glycols as hydrophilic structures and four C8 or four C18 alkyl chains as hydrophobic moieties into the aza-fluoro-boron fluorescent structure through slow water addition or flash precipitation. These nanostructures have very strong absorption at 808nm and have excellent photothermal conversion effect. The photothermal conversion efficiency of C8-NBDP-OEG4 nanoparticles can reach 39.8%, and can be used for photothermal treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1It is the structural formula of amphiphilic aza-fluoro-boron fluorescent dye;

[0045] Figure 2 The synthetic route diagram of amphiphilic aza-fluoro-boron fluorescent dyes;

[0046] Figure 3 This is the synthesis circuit diagram of C8-NBDP-OEG4;

[0047] Figure 4 C 18 - Synthesis circuit diagram of NBDP-OEG4;

[0048] Figure 5 is the H NMR spectrum of compound A;

[0049] Figure 6 is the NMR carbon spectrum of compound A;

[0050] Figure 7 is the H NMR spectrum of compound B;

[0051] Figure 8 is the NMR carbon spectrum of compound B;

[0052] Fig. 9 is the H NMR spectrum of compound C;

[0053] Fig.10 is the C NMR spectrum of compound C;

[0054] Fig.11 is the H NMR spectrum of compound D;

[0055] Fig.12 is the NMR carbon spectrum of compound D;

[0056] Fig.13 is the H NMR spectrum of compound C8-NBDP-OEG4;

[0057] Fig.14 is the NMR carbon spectrum of compound C8-NBDP-OEG4;

[0058] Fig.15 is the H NMR spectrum of compound G;

[0059] Fig.16 is the high-resolution mass spectrum of compound C;

[0060] Fig.17 is the high-resolution mass spectrum of compound D;

[0061] Fig.18 This is the high-resolution mass spectrum of compound C8-NBDP-OEG4;

[0062] Fig.19 C 18-High-resolution mass spectrometry of NBDP-OEG4;

[0063] Fig. 20 Absorption and emission spectra of compound C8-NBDP-OEG4 in different solvents;

[0064] Fig.21 (a) Absorption spectrum of C8-NBDP-OEG4 nanoparticles in PBS; (b) TEM image of C8-NBDP-OEG4 nanoparticles; (c) Particle size distribution of C8-NBDP-OEG4 nanoparticles;

[0065] Fig. 22 Different assembly morphologies obtained by assembling C18-NBDP-OEG4, (a) is a nanosheet; (b) is a branched nanorod structure;

[0066] Fig.23 (a) Different concentrations of C8-NBDP-OEG4 nanoparticles at 2.0 W / cm 2 Photothermal effect diagram under laser power; (b) Photothermal effect diagram of 0.05g / L C8-NBDP-OEG4 nanoparticles at different laser powers at 808nm; (c) Photothermal effect diagram of 0.05g / L C8-NBDP-OEG4 nanoparticles at 1.8W / cm 2 Heating-cooling curve under laser power irradiation; (d) Linear relationship between -ln(θ) and illumination time; (e) Photothermal cycle curve of C8-NBDP-OEG4 nanoparticles and ICG; (f) UV absorption spectrum of C8-NBDP-OEG4 nanoparticles before and after 808nm laser irradiation;

[0067] Fig.24 a) The cell survival rate after the cells were treated with different concentrations of C8-NBDP-OEG4 nanoparticles; b) The cell staining results after the cells were treated with 200 μg / mL C8-NBDP-OEG4 nanoparticles and without. DETAILED DESCRIPTION

[0068] The present invention is described in detail below with reference to the embodiments.

[0069] Example 1

[0070] An amphiphilic aza-fluoro-boron fluorescent dye C8-NBDP-OEG, whose structural formula is:

[0071]

[0072] The synthetic route of C8-NBDP-OEG4 is as follows:

[0073]

[0074] The preparation method of C8-NBDP-OEG4 is as follows:

[0075] Synthesis of compound A: p-Aminobenzaldehyde (3.64 g, 30 mmol, 1.0 equiv.) and 1-bromooctane (12.68 g, 66 mmol, 2.2 equiv.) were dissolved in 30 mL of DMF, and DIPEA (8.53 g, 66 mmol, 2.2 equiv.) was added thereto. The mixture was reacted at 110 ° C. for 12 h under a nitrogen atmosphere, and then returned to room temperature. The solvent was removed under reduced pressure, and the mixture was washed with water and subjected to column chromatography. The eluent for column chromatography was a mixed solution of PE:DCM in a volume ratio of 1:1. Finally, 11.5 g of product was obtained with a yield of 91.6%. 1 H NMR(400MHz,Chloroform-d)δ9.70(s,1H),7.70(d,J=8.5Hz,2H),6.64(d,J=8.6Hz, 2H),3.38–3.30(m,4H),1.60(q,J=7.2Hz,4H),1.27(s,20H),0.89(t,J=6.5Hz,6H). 13 CNMR(101MHz,Chloroform-d)δ190.13,152.77,132.43,110.97,51.39,32.02,29.64,29.50,27.36,27.28,22.87,14.32.MALDI-TOF-MS(m / z):Calcd.for[M+H] + ,346.5698,found,346.3397. Its H NMR spectrum and C NMR spectrum are as follows: Figure 5 , Figure 6 shown.

[0076] Synthesis of compound B: p-Hydroxyacetophenone (1.36 g, 10.0 mmol, 1.0 equiv.), tetraethylene glycol mono-p-toluenesulfonate (4.0 g, 11 mmol, 1.1 equiv.), and N,N-diisopropylethylamine (2.58 g, 20 mmol, 2.0 equiv.) were dissolved in 20 mL of anhydrous DMF. The mixture was reacted at 110 ° C for 6 h under a nitrogen atmosphere and then returned to room temperature. The DMF was removed under reduced pressure, and 50 mL of dichloromethane was added to dissolve the mixture. The mixture was washed three times with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, concentrated, and then subjected to column chromatography. The eluent for column chromatography was a mixed solution of dichloromethane: methanol in a volume ratio of 95:5 to obtain 2.9 g of an oily product with a yield of 89%. 1H NMR(400MHz,Chloroform-d)δ8.04–7.80(m,2H),7.05–6.87(m,2H),4.19(t,J=4.9Hz,2H),3.90–3. 86(m,2H),3.73–3.62(m,10H),3.54(t,J=4.5Hz,2H),3.37(d,J=1.3Hz,3H),2.55(d,J=1.5Hz,3H). 13 C NMR (101MHz, Chloroform-d)δ144.93,133.07,129.96,128.06,128.03,77.07,72.00,70.77,70.66,70.59,69.43,68.73,59.08,21.73. Its H NMR spectrum and C NMR spectrum are as follows: Figure 7 , Figure 8 shown.

[0077] Synthesis of compound C: Compound A (1.72 g, 5 mmol, 1.0 equiv.), compound B (1.63 g, 5 mmol, 1.0 equiv.) and sodium hydroxide (400 mg, 10 mmol, 2.0 equiv.) were dissolved in 20 mL of ethanol, stirred at room temperature for 12 h, concentrated to remove ethanol, dissolved in ethyl acetate, washed with saturated brine until neutral, the organic phase was dried over anhydrous sodium sulfate, concentrated and subjected to column chromatography. The eluent for column chromatography was a mixed solution of dichloromethane: methanol in a volume ratio of 95:5 to obtain 2.4 g of a yellow oily product with a yield of 74%. 1 H NMR (400MHz, Chloroform-d) δ8.07–7.97 (m, 2H), 7.73-7.80 (d, J = 15.4Hz, 1H), 7.51 (d, J = 8. 4Hz,2H),7.32(d,J=15.4Hz,1H),6.99–6.95(m,2H),6.62(d,J=8.5Hz,2H),4.20(t,J=4.9Hz ,2H),3.89(dd,J=3.8,2.3Hz,2H),3.67(tdd,J=7.7,3.6,2.1Hz,10H),3.57–3.54(m,2H),3. 30(d,J=7.8Hz,3H),1.61(d,J=9.4Hz,6H),1.30(dd,J=15.5,5.2Hz,22H),0.96–0.81(m,6H). 13C NMR(101MHz,Chloroform-d)δ150.15,145.29,130.76,130.65,116.17,114.48,114.45,111.54,72.15,71.10,70.85,70.83,70.74 ,69.80,69.74,67.84,67.80,59.25,51.27,32.04,29.68,29.52,27.50,27.33,22.87,14.33.MALDI-TOF-MS(m / z):Calcd.for[M+H] + ,654.9393,found,654.5025. Its H NMR spectrum, C NMR spectrum and mass spectrum are as follows: Fig. 9 , Fig.10 , Fig.16 shown.

[0078] Synthesis of compound D: Compound C (2.0 g, 3.06 mmol, 1.0 equiv.) was dissolved in 20 mL of ethanol, and nitromethane (434 mg, 7.12 mmol, 2.0 equiv.) and sodium hydroxide (122.4 mg, 3.06 mmol, 1.0 equiv.) were added thereto. After reacting at 70 ° C for 6 h, ethanol was removed under reduced pressure, and the mixture was dissolved in ethyl acetate and washed twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate, concentrated and subjected to column chromatography. The eluent for column chromatography was a mixed solution of dichloromethane: methanol in a volume ratio of 95:5 to obtain 1.2 g of a slightly yellow oily product with a yield of 55%. 1 H NMR(400MHz,Chloroform-d)δ7.89(d,J=8.5Hz,2H),7.07(d,J=8.3Hz,2H),6.93(d,J=8.6Hz,2H), 6.54(d,J=8.3Hz,2H),4.81-4.51(m,2H),4.18(t,J=4.8Hz,2H),4.11-4.02(m,1H),3.88(q,J=4.7, 4.0Hz,2H),3.73(t,J=3.3Hz,2H),3.67(td,J=14.7,14.1,7.4Hz,10H),3.54(dd,J=5.9,3.6Hz,2H) ,3.20(t,J=7.7Hz,3H),1.54(q,J=7.5Hz,5H),1.29(dd,J=7.7,4.2Hz,21H),0.88(t,J=6.6Hz,6H). 13C NMR(101MHz,Chloroform-d)δ147.85,138.75,130.52,128.39,125.27,114.60,111.96,80.32,72.16,71.11,70.84,70. 75,69.71,67.88,59.26,51.23,41.74,32.05,29.71,29.55,27.40,22.87,14.33.MALDI-TOF-MS(m / z):Calcd.for[M+H] + ,715.9793,found,715.5202. Its H NMR spectrum, C NMR spectrum and mass spectrum are as follows: Fig.11 , Fig.12 , Fig.17 shown.

[0079] Synthesis of compound C8-NBDP-OEG4: Compound D (714 mg, 1.0 mmol, 1.0 equiv.) was dissolved in 20 mL of n-butanol, and ammonium acetate (770 mg, 10.0 mmol, 10.0 equiv.) was added thereto. The mixture was reacted at 110 °C for 12 h under a nitrogen atmosphere, and then returned to room temperature. The n-butanol was removed by concentration, and the mixture was dissolved in dichloromethane and washed twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain an oily product which was directly used for the next step. The obtained product was dissolved in 20 mL of anhydrous dichloromethane. Under a nitrogen atmosphere, N,N-diisopropylethylamine (387 mg, 3.0 mmol, 3.0 equiv.) was slowly added thereto at -20°C. After stirring for 10 min, boron trifluoride etherate (710 mg, 5.0 mmol, 5.0 equiv.) was slowly added. After stirring for 10 min, the mixture was returned to room temperature and reacted for another 12 h. The mixture was added to 50 mL of saturated brine and washed twice. The organic phase was dried over anhydrous sodium sulfate, concentrated and subjected to column chromatography. The eluent for column chromatography was a mixed solution of dichloromethane: methanol in a volume ratio of 95:5. Finally, 110 mg of a viscous product was obtained with a yield of 15.8%. 1 HNMR(500MHz,Chloroform-d)δ8.04(dd,J=22.4,8.6Hz,6H),7.45(m,2H),6.97(dd,J=17.7,8.3Hz,4H),6.78(s,2H),6.69(d,J=8.6 Hz,4H),4.19(q,J=5.0Hz,4H),3.89(t,J=4.8Hz,6H),3.81–3.45(m,30H),3.38(s,6H),1.39–1.24(m,48H),0.89(t,J=6.7Hz,12H).13 C NMR(126MHz,Chloroform-d)δ160.68,156.87,149.13,145.51,143.23,131.46, 131.36,125.61,120.82,114.87,114.71,114.61,111.76,72.30,71.22,71.01, 70.89,70.00,70.00,67.79,59.42,51.53,32.23,30.53,30.08,29.91,29.87,2 9.76,27.82,27.63,27.57,23.05,14.50.MALDI-TOF-MS(m / z):Calcd.for[M+H] + ,1390.96,found,1390.0263. Its H NMR spectrum, C NMR spectrum and mass spectrum are as follows: Fig.13 , Fig.14 , Fig.18 shown.

[0080] The absorption and emission spectra of C8-NBDP-OEG4 in different solvents are shown in Fig. 20 As shown in the figure, it can be seen that C8-NBDP-OEG4 has very strong absorption between 600-900nm, is less affected by the solvent, and the fluorescence emission peak position is after 900nm, with good near-infrared second zone luminescence characteristics.

[0081] Example 2

[0082] An amphiphilic aza-fluoro-boron fluorescent dye C 18 -NBDP-OEG, its structural formula is:

[0083]

[0084] C 18 The synthetic route of -NBDP-OEG4 is as follows:

[0085]

[0086] C 18 -NBDP-OEG4 is prepared as follows:

[0087] Synthesis of compound E: p-Aminobenzaldehyde (1.82 g, 15 mmol, 1.0 equiv.) and 1-bromooctadecane (11.0 g, 33 mmol, 2.2 equiv.) were dissolved in 30 mL of DMF, and DIPEA (4.265 g, 33 mmol, 2.2 equiv.) was added thereto. The mixture was reacted at 110 ° C. for 12 h under a nitrogen atmosphere, and then returned to room temperature. The solvent was removed under reduced pressure, and the mixture was washed with water and subjected to column chromatography. The eluent for column chromatography was a mixed solution of PE:DCM in a volume ratio of 1:1. Finally, 7.2 g of product was obtained with a yield of 83.5%. 1 HNMR(400MHz,Chloroform-d)δ7.20(d,J=8.5Hz,2H),6.630(d,J=8.6Hz,2H),3.24(m,4H),1.56(q,J=7.2Hz,4H),1.27(s,60H),0.890(t,J=6.5Hz,6H).

[0088] Synthesis of compound F: Compound E (2.92 g, 5 mmol, 1.0 equiv.), compound B (1.63 g, 5 mmol, 1.0 equiv.) and sodium hydroxide (400 mg, 10 mmol, 2.0 equiv.) were dissolved in 20 mL of ethanol, stirred at room temperature for 12 h, concentrated to remove ethanol, dissolved in ethyl acetate, washed with saturated brine until neutral, the organic phase was dried over anhydrous sodium sulfate, concentrated and subjected to column chromatography, the eluent for column chromatography was a mixed solution of dichloromethane: methanol in a volume ratio of 95:5, to obtain 2.98 g of a yellow oily product with a yield of 68%. The obtained product was directly subjected to the next step reaction.

[0089] Synthesis of compound G: Compound C (2.5 g, 2.85 mmol, 1.0 equiv.) was dissolved in 20 mL of ethanol, and nitromethane (347 mg, 5.7 mmol, 2.0 equiv.) and sodium hydroxide (114 mg, 2.85 mmol, 1.0 equiv.) were added thereto. After reacting at 70 ° C for 6 h, ethanol was removed under reduced pressure, dissolved in ethyl acetate, washed twice with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, concentrated, and then subjected to column chromatography. The eluent for column chromatography was a mixed solution of dichloromethane: methanol in a volume ratio of 95:5 to obtain 1.4 g of a slightly yellow oily product with a yield of 52.3%. 1 H NMR (400 MHz, Chloroform-d) 1H NMR (400MHz, Chloroform-d) δ7.89 (d, J = 8.5Hz, 2H), 7.07 (d, J = 8.3Hz, 2H), 6.93 (d, J = 8.6Hz, 2H), 6.54 (d, J = 8.3Hz, 2H), 4.81-4.51 (m, 2H), 4.18 (t, J = 4.8Hz, 2H), 3.88 (q, J = 4.7, 4.0Hz, 2H), 3.75-3.50 (m, 12H), 3.40-3.16 (m, 8H), 1.54 (q, J = 7.5Hz, 4H), 1.29 (dd, J = 7.7, 4.2Hz, 60H), 0.88 (t, J = 6.6Hz, 6H). Its H NMR spectrum is as follows Fig.15 shown.

[0090] Compound C 18 - Synthesis of NBDP-OEG4: Compound G (938 mg, 1.0 mmol, 1.0 equiv.) was dissolved in 20 mL of n-butanol, and ammonium acetate (770 mg, 10.0 mmol, 10.0 equiv.) was added thereto. The mixture was reacted at 110 °C for 12 h under a nitrogen atmosphere, and then returned to room temperature. The n-butanol was removed by concentration, and the mixture was dissolved in dichloromethane and washed twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated to obtain an oily product for the next step. The obtained product was dissolved in 20 mL of anhydrous dichloromethane. Under a nitrogen atmosphere, N,N-diisopropylethylamine (387 mg, 3.0 mmol, 3.0 equiv.) was slowly added thereto at -20°C. After stirring for 10 min, boron trifluoride ether (710 mg, 5.0 mmol, 5.0 equiv.) was slowly added. After stirring for 10 min, the mixture was returned to room temperature and reacted for another 12 h. The mixture was added to 50 mL of saturated saline and washed twice. The organic phase was dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography. The eluent for column chromatography was a mixed solution of dichloromethane and methanol in a volume ratio of 95:5. Finally, 110 mg of a dark brown solid product was obtained with a yield of 12%. MALDI-TOF-MS (m / z): Calcd. for [M+H] + ,1949.57,found,1948.5347. Its mass spectrum is as follows Fig.19 shown.

[0091] Example 3

[0092] Preparation of C8-NBDP-OEG4 nanoparticles

[0093] 2 mg of C8-NBDP-OEG4 was dissolved in 1 mL of DMSO and quickly added into 9 mL of deionized water stirred at 1500 r / min under 4% CO. After stirring for 10 min, it was dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 3000 Da. The water was changed every 4 h and the obtained nanoparticles were placed in a refrigerator for later use.

[0094] The absorption spectrum of C8-NBDP-OEG4 nanoparticles in PBS is shown in Fig.21 As shown in Figure a), it can be seen from the figure that the nanoparticles have absorption in the range of 600-900nm, and the absorption at 808nm is very strong, which is suitable for subsequent photothermal test characterization.

[0095] TEM images of C8-NBDP-OEG4 nanoparticles Fig.21 As shown in Figure b), it can be seen from the figure that it is nanoparticles with uniform particle size.

[0096] The particle size distribution of C8-NBDP-OEG4 nanoparticles is shown in Fig.21 As shown in Figure c), it can be seen from the figure that the average particle size is 91nm.

[0097] Example 4

[0098] C 18 Preparation of -NBDP-OEG4 nanosheets

[0099] 0.5mg C 18 -NBDP-OEG4 was dissolved in 1 mL of DMF, 9 mL of deionized water was slowly added at a rate of 2 mL / h, and stirring was continued for 10 min. Then, the mixture was dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 3000 Da, and the water was changed every 4 h to obtain a uniform nanosheet structure. Fig. 22 As shown in Figure a), it can be seen from the figure that it is a nanosheet structure.

[0100] Example 5

[0101] C 18 - Preparation of NBDP-OEG4 branched nanorods:

[0102] 0.5mg C 18 -NBDP-OEG4 was dissolved in 1 mL of DMSO, and 9 mL of deionized water was slowly added at a rate of 1 mL / h. After stirring for 10 min, the mixture was dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 3000 Da, and the water was changed every 4 h to obtain a branched nanorod structure. Fig. 22 As shown in Figure b), it can be seen from the figure that it is a branch-like nanorod structure.

[0103] Example 6

[0104] Photothermal experiment of C8-NBDP-OEG4

[0105] C8-NBDP-OEG4 nanoparticles with different concentrations (0.2 g / L, 0.1 g / L, 0.05 g / L) were used with a rated power (2.5 W / cm 2 ) was irradiated with an 808nm laser and the temperature was recorded every 30s. After the temperature reached equilibrium, the laser was removed and the temperature change was recorded. The results are shown in Fig.23 As shown in Figure (a), it can be seen that under the same irradiation time, the higher the concentration of C8-NBDP-OEG4 nanoparticles, the higher the temperature, and regardless of the concentration of C8-NBDP-OEG4 nanoparticles, the temperature increases with the laser irradiation time. It can be seen that C8-NBDP-OEG4 nanoparticles have excellent photothermal responsiveness.

[0106] C8-NBDP-OEG4 nanoparticles with a concentration of 0.05 g / L were heated at different powers (0.38, 1.0, 1.5, 1.8, 2.5 W / cm 2 ) was irradiated with an 808nm laser and the temperature was recorded every 30s. After the temperature reached equilibrium, the laser was removed and the temperature change was recorded. The results are shown in Fig.23 As shown in Figure (b), under the same irradiation time, the higher the laser power, the higher the temperature, and no matter what the laser power is, the temperature increases with the laser irradiation time. It can be seen that C8-NBDP-OEG4 nanoparticles have excellent photothermal responsiveness.

[0107] 0.05 g / L C8-NBDP-OEG4 nanoparticles at 1.8 W / cm 2 The temperature rise-cooling curve under laser power irradiation is as follows Fig.23 As shown in Figure (c), when the temperature rises to the highest temperature of 66°C, the laser is removed and the temperature slowly returns to room temperature of 21°C. The photothermal conversion efficiency is calculated by the following formula:

[0108]

[0109] Where Tmax is 66℃; Tsurr is 21℃; A is the sample absorption value at 808nm, which is 1.06; I is the laser power, which is 1.8W / cm 2 ;Q Dis It represents the heat emitted by the light absorbed by the quartz sample cell itself, and the value is 30mW; hA is calculated by the following formula:

[0110]

[0111] Where m D The sample mass is 1.0 g, C D is the heat capacity (4.2 J / g), τ s It can be calculated using the following formula:

[0112]

[0113] Where t is time, unit is min; T RT is the temperature at each time point, T max and T surr and the highest temperature and room temperature respectively, so the τ s The calculation results are as follows Fig.23 As shown in d, combined with the above formula, the final photothermal conversion efficiency is 39.8%.

[0114] Fig.23 Figure (e) shows the temperature rise and fall cycle process of C8-NBDP-OEG4 nanoparticles after laser irradiation. Compared with the commercial dye ICG, it can be seen from the figure that C8-NBDP-OEG4 has good photostability. The highest temperature did not drop significantly during several temperature rise and fall cycles, while the photothermal effect of the commercial dye ICG decreased significantly after several irradiations. Fig.23 The ultraviolet absorption spectrum in Figure (f) also shows that the absorption of C8-NBDP-OEG4 does not change significantly after several temperature increases and decreases.

[0115] Example 7

[0116] Photothermal therapy at the cellular level

[0117] HeLa cells were seeded in 96-well plates at an initial density of 5000 / well (six parallel groups were set up) and incubated in a 37°C incubator with 5% CO2 for 24 h. Then, different concentrations of C8-NBDP-OEG4 nanoparticles (0, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL) were added. After incubation for 12 h, the old culture medium was removed and replaced with new culture medium. The culture medium was illuminated by an 808 nm laser (1.0 W / cm 2 ) After 5 minutes of illumination, the cells were incubated for another 4 hours, and then 20 μL of 5 mg / mL MTT solution was added. After 4 hours of incubation, the solution was removed, and then 200 μL of DMSO solution was added. After oscillation for 15 minutes, the absorption wavelength at 490 nm was measured with an ELISA reader, and the survival rate of the cells in each group of experiments was calculated. The results are shown in Fig.24As shown in the figure a), laser (+) indicates the laser irradiation group, and laser (-) indicates no laser irradiation. It can be seen from the figure that with the increase of concentration, the cell survival rate of the laser irradiation group decreased significantly, while there was no obvious apoptosis in the cells of the no laser irradiation group. 2,7-dichlorofluorescein diethyl ester was used to stain the living cells, and propidium iodide was used to stain the dead cells. The fluorescence confocal microscope was used for observation. The green fluorescence channel was excited with a 488nm laser and collected at 500-530nm. The red fluorescence channel was excited with 543 and the collection wavelength was 580-630nm. It can be seen that the cells in the laser irradiation group were all stained red, indicating that they contained a large number of dead cells, while the no laser irradiation group was green, indicating that they were all living cells.

[0118] The detailed description of an amphiphilic nitrogen-fluorine-boron fluorescent dye and its preparation method and application with reference to the above-mentioned embodiment is illustrative rather than restrictive, and several embodiments can be listed according to the limited scope. Therefore, changes and modifications without departing from the overall concept of the present invention should fall within the scope of protection of the present invention.

Claims

1. An amphiphilic aza-fluoro-boron fluorescent dye, characterized in that: The structural formula of the amphiphilic aza-fluoro-boron fluorescent dye is as follows: Wherein, R is R1 or R2; The structural formula of R1 is: This amphiphilic aza-fluoro-boron fluorescent dye is named C8-NBDP-OEG4; The structural formula of R2 is: This amphiphilic aza-fluoro-boron fluorescent dye is named C 18 -NBDP-OEG4.

2. The amphiphilic aza-fluoro-boron fluorescent dye according to claim 1, characterized in that: The C8-NBDP-OEG4 can be assembled into nanoparticles in water.

3. The amphiphilic aza-fluoro-boron fluorescent dye according to claim 1, characterized in that: The C 18 -NBDP-OEG4 can assemble into nanosheets or branched nanorods in water.

4. The method for preparing the amphiphilic aza-fluoro-boron fluorescent dye according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: 1) subjecting p-aminobenzaldehyde to a halogenation reaction with an alkyl halide to obtain an intermediate I; 2) subjecting p-hydroxyacetophenone to a nucleophilic substitution reaction with tetraethylene glycol mono-p-toluenesulfonate to obtain intermediate II; 3) reacting intermediate I and intermediate II under the catalysis of a base to obtain intermediate III; 4) Intermediate III reacts with nitromethane under the catalysis of a base to obtain intermediate IV; 5) Intermediate IV is subjected to coupling reaction with acetamide, and the obtained product is then reacted with boron trifluoride ether to obtain an amphiphilic aza-fluoro-boron fluorescent dye; The structural formula of the intermediate I is: The structural formula of the intermediate II is: The structural formula of the intermediate III is: The structural formula of the intermediate IV is:

5. The preparation method according to claim 4, characterized in that: The preparation method comprises the following steps: 1) dissolving p-aminobenzaldehyde and alkyl halide in DMF, adding DIPEA thereto, reacting at 105-115° C. for 10-12 h under nitrogen atmosphere, and purifying by column chromatography to obtain intermediate I; 2) dissolving p-hydroxyacetophenone, tetraethylene glycol mono-p-toluenesulfonate and N,N-diisopropylethylamine in anhydrous DMF, reacting at 105-115° C. for 5-6 hours under a nitrogen atmosphere, and purifying by column chromatography to obtain intermediate II; 3) Dissolve intermediate I, intermediate II and sodium hydroxide in ethanol, stir at room temperature for 10 to 12 hours, and purify by column chromatography to obtain intermediate III; 4) Intermediate III, nitromethane and sodium hydroxide are dissolved in ethanol, reacted at 65-75° C. for 5-6 hours, and purified by column chromatography to obtain intermediate IV; 5) Intermediate IV and acetamide are dissolved in n-butanol, and the reaction is carried out at 105-115° C. for 10-12 h in a nitrogen atmosphere. The product is dissolved in anhydrous dichloromethane, and N,N-diisopropylethylamine is slowly added thereto at -15--25° C. in a nitrogen atmosphere. After stirring evenly, boron trifluoride etherate is slowly added thereto, and the reaction is stirred at room temperature for 10-12 h. The product is purified by column chromatography to obtain an amphiphilic aza-fluoroboron fluorescent dye.

6. The preparation method according to claim 5, characterized in that: In step 1), the molar ratio of p-aminobenzaldehyde, alkyl halide and DIPEA is 1:2-5:2-5; the concentration of p-aminobenzaldehyde in DMF is 0.5-1.5M; And / or, in step 2), the molar ratio of p-hydroxyacetophenone, tetraethylene glycol mono-p-toluenesulfonate, and N,N-diisopropylethylamine is 1:1-1.5:2-2.5; the concentration of p-hydroxyacetophenone in anhydrous DMF is 0.1-1.0 M; And / or, in step 3), the molar ratio of intermediate I, intermediate II, and sodium hydroxide is 1:1-1.2:2-4; the concentration of intermediate I in ethanol is 0.1-0.5M; And / or, in step 4), the molar ratio of intermediate III, nitromethane and sodium hydroxide is 1:2-10:0.5-2.0; the concentration of intermediate III in ethanol is 0.1-0.5M; And / or, in step 5), the molar ratio of intermediate IV, acetic acid amine, N,N-diisopropylethylamine, and boron trifluoride etherate is 1:10-20:2-4:5-5.5; and the concentration of intermediate IV in n-butanol is 0.01-0.1M.

7. A method for preparing the amphiphilic aza-fluoro-boron fluorescent dye self-assembled nanoparticles according to claim 1, characterized in that: C8-NBDP-OEG4 was dissolved in dimethyl sulfoxide, which was quickly added into deionized water with high-speed stirring, and then the organic solvent was removed by dialysis to obtain uniform nanoparticles.

8. A method for preparing the amphiphilic aza-fluoro-boron fluorescent dye self-assembled nanosheets according to claim 1, characterized in that: C 18 -NBDP-OEG4 is dissolved in N,N-dimethylformamide, deionized water is slowly added thereto, and then the organic solvent is removed by dialysis to obtain uniform nanosheets.

9. A method for preparing self-assembled branched nanorods of amphiphilic aza-fluoro-boron fluorescent dyes according to claim 1, characterized in that: C 18 -NBDP-OEG4 was dissolved in dimethyl sulfoxide, deionized water was slowly added thereto, and then the organic solvent was removed by dialysis to obtain branched nanorods.

10. Use of the amphiphilic aza-fluoro-boron fluorescent dye according to claim 1 as a photothermal agent.

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