Preparation method of near-infrared two-region fluorescently-labeled phospholipid molecule and preparation method of lipid nano-carrier of phospholipid molecule

Abstract

The invention discloses a preparation method of a near-infrared two-region fluorescently-labeled phospholipid molecule and a preparation method of a lipid nano-carrier of the phospholipid molecule, and relates to the field of preparation methods of cyanine dyes. The invention aims to solve the technical problems that the application of the organic cyanine dye in a living body is limited by the hydrophobicity of the organic cyanine dye and the fluorescence intensity is low due to the aggregation quenching effect of the organic cyanine dye. The method comprises the following steps: preparing a compound 2 from 1, 8-naphthalimide and iodohexane, and then preparing C6 from the compound 2, 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-formaldehyde and sodium acetate; then C6 and mercaptobenzoic acid are used for preparing C6-COOH; the preparation method comprises the following steps: preparing C6-Lipid from N, N-diisopropylethylamine, HBTU, C6-COOH and 4-(1, 2, 2-triphenylvinyl) aniline by virtue of a hydrothermal method; the preparation method comprises the following steps: preparing a lipid nano-carrier from C6-Lipid, DMPC (Dimethyl Pyrrolidone), DOPE (Dioctyl Olefin Polyethylene) and According to the phospholipid molecule and the lipid nano-carrier thereof prepared by the invention, effective imaging of fluorophores is realized, and aggregates such as J aggregation and the like are possibly generated, so that the fluorescence quantum yield is improved, and an efficient fluorescence imaging effect is achieved. The cyanine dye prepared by the invention is used for fluorescence imaging.

Description

Technical Field

[0001] The invention relates to the field of preparation methods of cyanine dyes. Background Art

[0002] With the improvement of treatment precision and the reduction of cancer mortality, the development of near-infrared (NIR-I and NIR-II) phototherapy diagnostic systems has become increasingly important. These systems operate in the NIR-II wavelength range (1000–1700nm), which can significantly improve tissue penetration and imaging resolution while effectively avoiding the generation of autofluorescence, thus surpassing the performance of NIR-I wavelengths (700–900nm).

[0003] Compared with inorganic fluorescent groups, organic small molecule NIR-II organic cyanine dyes have attracted more and more attention due to their excellent tunable light physical properties, biocompatibility and minimal biological toxicity. Studies have shown that organic cyanine dyes with NIR-II absorption and emission wavelengths show good application potential in NIR-II fluorescence bioimaging. However, the hydrophobicity of organic cyanine dyes limits their application in vivo, and their aggregation quenching effect leads to low fluorescence intensity. Summary of the invention

[0004] The present invention provides a method for preparing phospholipid molecules labeled with near-infrared second-zone fluorescence in order to solve the technical problems that the hydrophobicity of organic cyanine dyes limits their use in organisms and their aggregation quenching effect leads to low fluorescence intensity.

[0005] A method for preparing a phospholipid molecule labeled with near-infrared second-zone fluorescence is specifically carried out in the following steps:

[0006] 1. Under a nitrogen atmosphere, 1,8-naphtholactimide and iodine hexane were dissolved in toluene, and heated under reflux at 100°C with stirring for 20 hours; after the reaction was completed, the mixture was cooled to room temperature, filtered, and washed with ethyl acetate to obtain compound 2;

[0007] 2. Add 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde, sodium acetate and compound 2 obtained in step 1 to acetic anhydride, react at 60° C. for 30 min, then cool, pour into saturated sodium bicarbonate solution, wash with brine, and dry to obtain C6;

[0008] 3. Dissolve mercaptobenzoic acid and C6 obtained in step 2 in a mixed solution of anhydrous dichloromethane and anhydrous methanol, stir at room temperature for 3 hours, and after the reaction is completed, obtain C6-COOH by thin layer chromatography;

[0009] 4. Dissolve N,N-diisopropylethylamine, HBTU and C6-COOH obtained in step 3 in anhydrous dichloromethane, stir at room temperature for 1 hour to obtain reaction solution 3; dissolve 4-(1,2,2-triphenylvinyl)aniline in anhydrous dichloromethane, add it to reaction solution 3, and stir at room temperature for 24 hours; after the reaction is completed, concentrate in vacuo, and purify by thin layer chromatography to obtain C6-Lipid.

[0010] The chemical formula of the C6-Lipid is:

[0011]

[0012] A method for preparing a phospholipid molecule labeled with near-infrared second-zone fluorescence is specifically carried out in the following steps:

[0013] Ⅰ. Under an argon atmosphere, sodium hydride and 1,8-naphtholactimide were dissolved in anhydrous N,N-dimethylformamide; then cooled to 0°C and added to iodohexane; stirred at room temperature for 3 hours. After the reaction was completed, extracted with ethyl acetate, washed with brine and concentrated, purified by silica gel column chromatography, and isolated to obtain compound 4;

[0014] II. Under argon atmosphere, the compound 4 obtained in step 7 was dissolved in anhydrous THF, methylmagnesium chloride was added, and the mixture was stirred at 55° C. for 1 h. After the reaction was completed, the reaction system was cooled and poured into an ice-water mixture containing HCl solution. THF was removed under reduced pressure, and the aqueous solution of the mixture was filtered. Then, potassium iodide solution was added to obtain a red precipitate. After the solvent was dried under vacuum, potassium iodide solution was added to obtain a red precipitate. The crude product was filtered, and the obtained precipitate was washed three times with ethyl acetate and water, and dried to obtain a solid product 5.

[0015] III. Add 2-chloro-3-(hydroxymethylene)-cyclohex-1-enecarboxaldehyde and the solid product 5 obtained in step II to 2 mL of acetic acid, and then add to a mixed solution of triethylamine and acetic anhydride, and react at 60° C. for 30 min; after the reaction is completed, cool, add ethyl acetate, filter, and dry to obtain NIR-C6;

[0016] IV. Dissolve the NIR-C6 obtained in step III and mercaptobenzoic acid in a mixed solution of anhydrous dichloromethane and anhydrous methanol, and stir at room temperature for 2 hours; after the reaction is completed, add to ice ether, filter, and obtain NIR-C6-COOH;

[0017] V. Dissolve the NIR-C6-COOH, N,N-diisopropylethylamine and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate obtained in step IV in anhydrous dichloromethane, stir at room temperature for 1 hour to obtain a reaction solution; distearoylphosphatidylethanolamine is dissolved in anhydrous dichloromethane, added to the reaction solution, and stirred at room temperature for 2 hours; after the reaction is completed, concentrate in vacuo, and purify by thin layer chromatography to obtain NIR-C6-COOH-Lipid.

[0018] The chemical formula of the NIR-C6-COOH-Lipid is:

[0019]

[0020] A method for preparing a lipid nanocarrier of a phospholipid molecule labeled with near-infrared second-zone fluorescence is specifically carried out in the following steps:

[0021] DMPC, DOPE, DSPE-PEG 2000 and C6-Lipid were dissolved in a mixed solution of chloroform and methanol in a volume ratio of 1:1; the mixed solution was evaporated at 30°C in a vacuum environment to form a uniform lipid film; then, the lipid film was dispersed in 1.0mL of phosphate solution and ultrasonicated on an ice bath for 2.5min to obtain a liposome preparation; the liposome preparation was subjected to three heating-cooling cycles between 50°C and 6°C, and then extruded back and forth 10 times through a polycarbonate membrane with a pore size of 400nm and a polycarbonate membrane with a pore size of 200nm using a small extruder, and then the liposomes were dialyzed through a dialysis bag to obtain C6-Lipid-L.

[0022] A method for preparing a lipid nanocarrier of a phospholipid molecule labeled with near-infrared second-zone fluorescence is specifically carried out in the following steps:

[0023] DMPC, DOPE, DSPE-PEG 2000 and NIR-C6-COOH-Lipid were dissolved in a mixed solution of chloroform and methanol in a volume ratio of 1:1; the mixed solution was evaporated under a vacuum environment at 30°C to form a uniform lipid film; then, the lipid film was dispersed in 1.0 mL of phosphate solution and ultrasonicated on an ice bath for 2.5 min to obtain a liposome preparation; the liposome preparation was subjected to three heating-cooling cycles between 50°C and 6°C, and then extruded back and forth 10 times through a polycarbonate membrane with a pore size of 400 nm and a polycarbonate membrane with a pore size of 200 nm using a small extruder, and then the liposomes were dialyzed through a dialysis bag to obtain NIR-C6-COOH-Lipid-L.

[0024] In the present invention, based on the synthesis of existing basic dyes, phospholipid-based NIR-I and NIR-II organic fluorophores C6-lipid and NIR-C6-COOH-lipid are further developed. This modification of phospholipids (components of liposomes) to the grafted parts of NIR-I and NIR-II will facilitate better self-assembly with other components of liposomes to achieve strong fluorescence emission capabilities.

[0025] Beneficial effects of the present invention:

[0026] The present invention provides a phospholipid molecule labeled with near-infrared second-zone fluorescence and a lipid nanocarrier thereof. The effective imaging of the fluorophore can be achieved by using similar phospholipid-modified organic fluorescent dyes, and aggregates such as J aggregation may be generated to improve the fluorescence quantum yield and achieve efficient fluorescence imaging effects.

[0027] The cyanine dye prepared by the invention is used for fluorescence imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Example Compound 2 in CDCl3 1 H NMR spectrum;

[0029] Figure 2 Example C6 in CDCl3 1 H NMR spectrum;

[0030] Figure 3 Example C6-COOH in CDCl3 1 H NMR spectrum;

[0031] Figure 4 Example C6-TPA in CDCl3 1 H NMR spectrum;

[0032] Figure 5 is a high-resolution mass spectrum of Example C6-TPA in CDCl3;

[0033] Figure 6 Example C6-TPE in CDCl3 1 H NMR spectrum;

[0034] Figure 7 is a high-resolution mass spectrum of Example C6-TPE;

[0035] Figure 8 Example C6-Lipid in CDCl3 1 H NMR spectrum;

[0036] Fig. 9 This is the high-resolution mass spectrum of Example C6-Lipid;

[0037] Fig.10 Example compound 4 in CDCl3 1 H NMR spectrum;

[0038] Fig.11 Example compound 5 in CDCl3 1 H NMR spectrum;

[0039] Fig.12 For example, NIR-C6 in CDCl3 1 H NMR spectrum;

[0040] Fig.13 For example, NIR-C6-COOH in CDCl3 1 H NMR spectrum;

[0041] Fig.14 This is the high-resolution mass spectrum of Example NIR-C6-COOH;

[0042] Fig.15 For example, NIR-C6-COOH-Lipid in CDCl3 1 H NMR spectrum;

[0043] Fig.16 This is the high-resolution mass spectrum of NIR-C6-COOH-Lipid in Example;

[0044] Fig.17 It is a normalized UV-visible-near infrared absorption spectrum of Example C6-TPA, C6-TPE and C6-Lipid in dichloromethane;

[0045] Fig.18 It is a normalized fluorescence emission spectra of Example C6-TPA, C6-TPE and C6-Lipid in dichloromethane;

[0046] Fig.19 is a normalized UV-visible-near infrared absorption spectrum of Example NIR-C6-COOH-Lipid in dichloromethane;

[0047] Fig. 20 It is a normalized UV-visible-near infrared absorption spectrum of Example C6-TPA-L, C6-TPE-L and C6-Lipid-L;

[0048] Fig.21 The fluorescence emission spectra of Examples C6-TPA-L, C6-TPE-L and C6-Lipid;

[0049] Fig. 22It is the near-infrared second-zone fluorescence imaging image of Example C6-TPA-L, C6-TPE-L and C6-Lipid-L;

[0050] Fig.23 It is the normalized UV-visible-near infrared absorption spectra of Examples NIR-C6-L and NIR-C6-COOH-Lipid-L;

[0051] Fig.24 It is the normalized fluorescence emission spectra of Example NIR-C6-L and NIR-C6-COOH-Lipid-L;

[0052] Fig.25 is a transmission electron micrograph of Example C6-Lipid-L;

[0053] Fig.26 is a particle size diagram of Example C6-Lipid-L;

[0054] Fig. 27 is a transmission electron microscopy image of Example NIR-C6-COOH-Lipid-L;

[0055] Fig.28 This is a dynamic light scattering curve diagram of Example NIR-C6-COOH-Lipid-L. DETAILED DESCRIPTION

[0056] Specific implementation method 1: This implementation method is a method for preparing a phospholipid molecule labeled with near-infrared second-region fluorescence, which is specifically carried out in the following steps:

[0057] 1. Under a nitrogen atmosphere, 1,8-naphtholactimide and iodine hexane were dissolved in toluene, and heated under reflux at 100°C with stirring for 20 hours; after the reaction was completed, the mixture was cooled to room temperature, filtered, and washed with ethyl acetate to obtain compound 2;

[0058] 2. Add 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde, sodium acetate and compound 2 obtained in step 1 to acetic anhydride, react at 60° C. for 30 min, then cool, pour into saturated sodium bicarbonate solution, wash with brine, and dry to obtain C6;

[0059] 3. Dissolve mercaptobenzoic acid and C6 obtained in step 2 in a mixed solution of anhydrous dichloromethane and anhydrous methanol, stir at room temperature for 3 hours, and after the reaction is completed, obtain C6-COOH by thin layer chromatography;

[0060] 4. Dissolve N,N-diisopropylethylamine, HBTU and C6-COOH obtained in step 3 in anhydrous dichloromethane, stir at room temperature for 1 hour to obtain reaction solution 3; dissolve 4-(1,2,2-triphenylvinyl)aniline in anhydrous dichloromethane, add it to reaction solution 3, and stir at room temperature for 24 hours; after the reaction is completed, concentrate in vacuo, and purify by thin layer chromatography to obtain C6-Lipid.

[0061] Specific embodiment 2: This embodiment is different from the specific embodiment 1 in that the usage ratio of 1,8-naphtholactimide, iodohexane and toluene in step 1 is 10 mmol: 20 mmol: 20 mL. The rest is the same as the specific embodiment 1.

[0062] Specific embodiment 3: This embodiment is different from specific embodiment 1 or 2 in that the usage ratio of acetic anhydride, 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde, sodium acetate and compound 2 in step 2 is 20mL:3.12mmol:3.43mmol:6.24mmol. The rest is the same as specific embodiment 1 or 2.

[0063] Specific embodiment 4: This embodiment differs from specific embodiments 1 to 3 in that the usage ratio of mercaptobenzoic acid, C6, anhydrous dichloromethane and anhydrous methanol in step 3 is 0.17mmol:0.083mmol:5mL:5mL. The rest is the same as specific embodiments 1 to 3.

[0064] Specific embodiment 5: This embodiment is different from specific embodiments 1 to 4 in that the usage ratio of N,N-diisopropylethylamine, HBTU, C6-COOH and 4-(1,2,2-triphenylvinyl)aniline in step 4 is 0.070mmol: 0.11mmol: 0.056mmol: 0.084mmol 4-(1,2,2-triphenylvinyl)aniline. The rest is the same as specific embodiments 1 to 4.

[0065] Specific embodiment 6: This embodiment is different from any one of the specific embodiments 1 to 5 in that the chemical formula of the C6-Lipid is:

[0066]

[0067] The rest is the same as that of the specific implementation modes 1 to 5.

[0068] Specific embodiment 7: This embodiment is a method for preparing a phospholipid molecule labeled with near-infrared second region fluorescence, which is specifically carried out according to the following steps:

[0069] Ⅰ. Under an argon atmosphere, sodium hydride and 1,8-naphtholactimide were dissolved in anhydrous N,N-dimethylformamide; then cooled to 0°C and added to iodohexane; stirred at room temperature for 3 hours. After the reaction was completed, extracted with ethyl acetate, washed with brine and concentrated, purified by silica gel column chromatography, and isolated to obtain compound 4;

[0070] II. Under argon atmosphere, the compound 4 obtained in step 7 was dissolved in anhydrous THF, methylmagnesium chloride was added, and the mixture was stirred at 55° C. for 1 h. After the reaction was completed, the reaction system was cooled and poured into an ice-water mixture containing HCl solution. THF was removed under reduced pressure, and the aqueous solution of the mixture was filtered. Then, potassium iodide solution was added to obtain a red precipitate. After the solvent was dried under vacuum, potassium iodide solution was added to obtain a red precipitate. The crude product was filtered, and the obtained precipitate was washed three times with ethyl acetate and water, and dried to obtain a solid product 5.

[0071] III. Add 2-chloro-3-(hydroxymethylene)-cyclohex-1-enecarboxaldehyde and the solid product 5 obtained in step II to 2 mL of acetic acid, and then add to a mixed solution of triethylamine and acetic anhydride, and react at 60° C. for 30 min; after the reaction is completed, cool, add ethyl acetate, filter, and dry to obtain NIR-C6;

[0072] IV. Dissolve the NIR-C6 obtained in step III and mercaptobenzoic acid in a mixed solution of anhydrous dichloromethane and anhydrous methanol, and stir at room temperature for 2 hours; after the reaction is completed, add to ice ether, filter, and obtain NIR-C6-COOH;

[0073] V. Dissolve the NIR-C6-COOH, N,N-diisopropylethylamine and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate obtained in step IV in anhydrous dichloromethane, stir at room temperature for 1 hour to obtain a reaction solution; distearoylphosphatidylethanolamine is dissolved in anhydrous dichloromethane, added to the reaction solution, and stirred at room temperature for 2 hours; after the reaction is completed, concentrate in vacuo, and purify by thin layer chromatography to obtain NIR-C6-COOH-Lipid.

[0074] Specific embodiment eight: This embodiment differs from specific embodiment seven in that the chemical formula of the NIR-C6-COOH-Lipid is:

[0075]

[0076] The rest is the same as the seventh specific implementation method.

[0077] Specific embodiment 9: This embodiment is a method for preparing a lipid nanocarrier of a phospholipid molecule labeled with near-infrared second-region fluorescence, which is specifically carried out according to the following steps:

[0078] DMPC, DOPE, DSPE-PEG 2000 and C6-Lipid were dissolved in a mixed solution of chloroform and methanol in a volume ratio of 1:1; the mixed solution was evaporated at 30°C in a vacuum environment to form a uniform lipid film; then, the lipid film was dispersed in 1.0mL of phosphate solution and ultrasonicated on an ice bath for 2.5min to obtain a liposome preparation; the liposome preparation was subjected to three heating-cooling cycles between 50°C and 6°C, and then extruded back and forth 10 times through a polycarbonate membrane with a pore size of 400nm and a polycarbonate membrane with a pore size of 200nm using a small extruder, and then the liposomes were dialyzed through a dialysis bag to obtain C6-Lipid-L.

[0079] Specific embodiment 10: This embodiment is a method for preparing a lipid nanocarrier of a phospholipid molecule labeled with near-infrared second-region fluorescence, which is specifically carried out in the following steps:

[0080] DMPC, DOPE, DSPE-PEG 2000 and NIR-C6-COOH-Lipid were dissolved in a mixed solution of chloroform and methanol in a volume ratio of 1:1; the mixed solution was evaporated under a vacuum environment at 30°C to form a uniform lipid film; then, the lipid film was dispersed in 1.0 mL of phosphate solution and ultrasonicated on an ice bath for 2.5 min to obtain a liposome preparation; the liposome preparation was subjected to three heating-cooling cycles between 50°C and 6°C, and then extruded back and forth 10 times through a polycarbonate membrane with a pore size of 400 nm and a polycarbonate membrane with a pore size of 200 nm using a small extruder, and then the liposomes were dialyzed through a dialysis bag to obtain NIR-C6-COOH-Lipid-L.

[0081] The content of the present invention is not limited to the content of the above-mentioned embodiments. The combination of one or several specific embodiments can also achieve the purpose of the invention.

[0082] Example:

[0083] The present embodiment provides a method for preparing a phospholipid molecule labeled with near-infrared second region fluorescence, which is specifically carried out in the following steps:

[0084] 1. Under nitrogen atmosphere, 10 mmol 1,8-naphtholactimide and 20 mmol iodohexane were dissolved in 20 mL toluene, and heated under reflux at 100°C with stirring for 20 h; after the reaction was completed, the mixture was cooled to room temperature, filtered, and washed with ethyl acetate to obtain a light pink solid product, compound 2 (2320 mg, 31.24%) 1H NMR(400MHz,Chloroform-d)δ8.38(d,J=14.1Hz,1H),7.42(d,J=7.4Hz,2H),7 .28(d,J=7.7Hz,1H),7.23–7.17(m,1H),6.26(d,J=14.0Hz,1H),4.22(t,J=7. 5Hz,2H),2.77(t,J=6.2Hz,2H),1.88(p,J=7.4Hz,2H),1.76(s,6H),1.49(d,J =8.1Hz,2H),1.38(dddd,J=15.3,7.9,6.1,3.8Hz,4H),0.92(t,J=6.9Hz,3H).;

[0085]

[0086] 2. Add 3.12 mmol 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde, 3.43 mmol sodium acetate and 6.24 mmol compound 2 obtained in step 1 to 20 mL acetic anhydride, react at 60°C for 30 min, then cool, pour into saturated sodium bicarbonate solution, wash with brine, and dry to obtain black solid C6 (1020 mg, yield 21.76%). 1 HNMR(400MHz,DMSO-d6)δ8.28(d,J=14.1Hz,2H),7.66(d,J=7.4Hz,2H),7.46(d,J=6.9Hz,4H),7.31(td,J=6.9,1.8Hz,2H),6.35(d,J=14.2Hz,2H), 4.24(t,J=7.4Hz,4H),2.73(t,J=6.2Hz,4H),1.92–1.82(m,2H),1.69(s,1 6H), 1.34 (dddd, J=19.4, 16.1, 13.8, 6.1Hz, 14H), 0.87 (t, J=6.9Hz, 6H).;

[0087]

[0088] 3. Dissolve 0.17mmol of mercaptobenzoic acid and 0.083mmol of C6 obtained in step 2 in a mixed solution of 5mL of anhydrous dichloromethane and 5mL of anhydrous methanol, and stir at room temperature for 3h. After the reaction is completed, thin layer chromatography (the volume ratio of dichloromethane to methanol is 10:1) is used to obtain black solid C6-COOH (22.4mg, yield 36.4%). 1H NMR (400MHz, DMSO-d6) δ8.58(d,J=14.1Hz,2H),7.84(d,J=8.1Hz,2H),7.55(d,J =7.5Hz,2H),7.41(d,J=5.7Hz,4H),7.34–7.19(m,4H),6.35(d,J=14.2Hz,2H),4. 19(t,J=7.3Hz,5H),2.79(t,J=6.2Hz,4H),1.96(p,J=6.8,6.3Hz,2H),1.71(p,J =7.2Hz,4H),1.39(d,J=16.7Hz,16H),1.31–1.23(m,9H),0.86(t,J=6.8Hz,6H).;

[0089]

[0090] 4. Dissolve 0.051mmol N,N-diisopropylethylamine (DIPEA), 0.11mmol benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and 0.040mmol C6-COOH obtained in step 3 in 3mL of anhydrous dichloromethane, stir at room temperature for 1h to obtain reaction solution 1; dissolve 0.061mmol N1,N1-diphenylbenzene-1,4-diamine in 1mL of anhydrous dichloromethane, then add to the reaction solution, and stir at room temperature for 3h; after the reaction is completed, concentrate in vacuo, and purify by column chromatography (the volume ratio of petroleum ether to tetrahydrofuran is 1:1) to obtain a black solid product C6-TPA (23.3mg, yield 58.6%) 1 H NMR (400MHz, DMSO-d6) δ10.15(s,1H),8.59(d,J=14.1Hz,2H),7.90(d,J=8.4Hz,2H),7.68– 7.64(m,2H),7.55(d,J=7.4Hz,2H),7.45–7.34(m,7H),7.30–7.20(m,7H),7.04–6.92(m,9H) ,6.36(d,J=14.2Hz,2H),4.19(t,J=7.5Hz,4H),3.66–3.46(m,1H),2.79(s,4H),1.80–1.64( m,5H),1.43(s,10H),1.32–1.22(m,19H),0.85(d,J=7.0Hz,6H).HRMS(ESI)m / z:calculated for C 67 H 75 N4OS + ,[M] +983.5656, found 983.5657.

[0091]

[0092] 5. Dissolve 0.084mmol DIPEA, 0.13mmol HBTU and 0.067mmol C6-COOH obtained in step 3 in 3mL anhydrous dichloromethane, stir at room temperature for 1h to obtain reaction solution 2; dissolve 0.101mmol 4-(1,2,2-triphenylvinyl)aniline in 1mL anhydrous dichloromethane, add to reaction solution 2, and stir at room temperature for 3h; after the reaction is completed, concentrate in vacuo, and purify by column chromatography (the volume ratio of dichloromethane to methanol is: 100:1) to obtain a black solid product C6-TPE (18.7 mg, yield 15.6%) 1 H NMR (400 MHz, DMSO-d6) δ10.10(s,1H),8.59(d,J=14.1 Hz,2H),7.88(d,J=8.4 Hz,2H),7.55(d,J=7.5 Hz,2H),7.49(d,J=8.6 Hz,3H),7.45–7.37(m,7H),7.26(ddd,J=8.1,6.1,2.3 Hz,2H),7.13(qd,J=7.9,6.8,3.8 Hz,12H),7.04–6.87(m,11H),6.36(d,J=14.2 Hz,2H),4.23–4.15(m,4H),3.53(s,1H),3.19(d,J=3.8 Hz,2H),2.80(s,4H),1.97(s,3H),1.70(q,J=7.4 Hz,5H),1.42–1.28(m,25H),0.86(d,J=6.9 Hz,6H).HRMS(ESI)m / z:calculated for C 75 H 80 N3OS+,[M+H]+1070.6017,found 1070.6017.;

[0093]

[0094] 6. Dissolve 0.070 mmol N,N-diisopropylethylamine, 0.11 mmol HBTU and 0.056 mmol C6-COOH obtained in step 3 in 3 mL of anhydrous dichloromethane, stir at room temperature for 1 h to obtain reaction solution 3; dissolve 0.084 mmol 4-(1,2,2-triphenylvinyl)aniline in 1 mL of anhydrous dichloromethane, add to reaction solution 3, and stir at room temperature for 24 h; after the reaction is completed, concentrate in vacuo and purify by thin layer chromatography (the volume ratio of dichloromethane to methanol is 20:1) to obtain a black solid product C6-Lipid (32.3 mg, yield 36.3%) 1 H NMR (400 MHz, CDCl3) δ9.94 (s, 1H), 8.72 (d, J = 14.1 Hz, 2H), 8.12 (d, J = 8.2 Hz, 2H), 7.41–7.32 (m, 5H), 7.28–7.18 (m, 4H), 7.07 (d, J = 7.9 Hz,2H),6.08(d,J=14.1 Hz,2H),5.33(s,1H),5.22(q,J=5.8,4.9 Hz,1H),4.43(dd,J=12.0,3.5 Hz,1H),4.26–4.04(m,6H),3.99(t,J=7.7 Hz,4H),3.58(q,J=4.2 Hz,2H),2.72(t,J=6.2 Hz,4H),2.29(td,J=7.6,3.1 Hz,4H),1.85(t,J=7.5Hz,4H),1.59(t,J=7.2 Hz,4H),1.46–1.18(m,87H),0.93(dd,J=17.0,6.6 Hz,12H).HRMS(ESI)m / z:calculated for C 90 H 141 N3O9PS+,[M+H]+1471.0121,found1471.0126.;

[0095]

[0096] A method for preparing a phospholipid molecule labeled with near-infrared second-zone fluorescence is specifically carried out in the following steps:

[0097] 7. Under argon atmosphere, 15 mmol sodium hydride (600 mg, 15 mmol, M = 24 g / mol, 60% dispersed in mineral oil) and 5 mmol 1,8-naphthoic acid imide were dissolved in 15 mL anhydrous N, N-dimethylformamide; then cooled to 0 ° C, and added to 0.88 mL iodohexane; stirred at room temperature for 3 h. After the reaction was completed, extracted with ethyl acetate, washed with brine and concentrated, and purified by silica gel column chromatography (the volume ratio of petroleum ether to ethyl acetate was 10: 1) to isolate the yellow liquid product compound 4 (980 mg, 77.4%). 1 H NMR (400MHz, CDCl3) δ8.06(d,J=6.9Hz,1H),7.99(d,J=8.1Hz,1H),7.73–7.67(m,1H),7.52(d,J=8.4Hz,1H),7.46(dd,J=8 .5,6.8Hz,1H),6.90(d,J=6.9Hz,1H),3.92(t,J=7.3Hz,2H),1.81(q,J=7.4Hz,2H),1.47–1.30(m,6H),0.97–0.82(m,3H).;

[0098]

[0099] 8. Under argon atmosphere, 3.86 mmol of compound 4 obtained in step 7 was dissolved in 5 mL of anhydrous THF, 5.02 mmol of methylmagnesium chloride was added, and the mixture was stirred at 55° C. for 1 h. After the reaction was completed, the reaction system was cooled and poured into 150 mL of an ice-water mixture containing 2 mL of a 32% HCl solution. THF was removed under reduced pressure, and the aqueous solution of the mixture was filtered. Then, potassium iodide solution was added to obtain a red precipitate. After the solvent was dried under vacuum, the crude product was filtered, and the precipitate was washed three times with ethyl acetate and water, and dried to obtain a red solid product 5 (732.02 mg, yield 50%). NMR (400MHz, CDCl3) δ8.99(d,J=7.2Hz,1H),8.64(d,J=8.1Hz,1H),8.41(d,J=7.4Hz, 1H), 8.31 (d, J = 8.2Hz, 1H), 8.09 (t, J = 7.7Hz, 1H), 7.93 (t, J = 7.8Hz, 1H), 4.89 (t, J = 7. 6Hz,2H),3.51(s,3H),2.05(p,J=7.7Hz,2H),1.73(s,1H),1.53(p,J=7.2Hz,2H),1.3 3(tdd,J=15.5,7.7,2.7Hz,4H),0.87(t,J=7.0Hz,3H).ESI-MSm / z[MI]calculatedfor C 18 H 22 N + 252.3, found 252.2;

[0100]

[0101] IX. 0.75 mmol 2-chloro-3-(hydroxymethylene)-cyclohex-1-enecarboxaldehyde and 1.5 mmol solid product 5 obtained in step 8 were added to 2 mL acetic acid, and then added to a mixed solution of 1.0 mL triethylamine and 1.0 mL acetic anhydride, and reacted at 60° C. for 30 min. After the reaction was completed, the mixture was cooled, 100 mL ethyl acetate was added, and a black precipitate was obtained by filtration, and dried to obtain NIR-C6 (402.80 mg, yield 35%). 1H NMR (400MHz, CDCl3) δ8.50(s,2H),8.15(s,2H),7.93(s,2H),7.85(t,J=7.7Hz,2H),7.48–7.37(m,4H),7.05(s,2H),6.43(s,2H),4.00(q,J =6.3Hz,4H),2.81(s,4H),2.07–2.01(m,2H),1.72(t,J=7.4Hz,4H),1.37(d,J=8.6Hz,4H),1.28–1.24(m,8H),0.84(d,J=6.6Hz,6H).ESI-MS m / z[MI]calculated forC 44 H 48 ClN2 + 639.3,found639.1.;

[0102]

[0103] 10. Dissolve 0.2 mmol of NIR-C6 obtained in step 9 and 0.4 mmol of mercaptobenzoic acid in a mixed solution of 10 mL of anhydrous dichloromethane and 10 mL of anhydrous methanol, and stir at room temperature for 2 h. After the reaction is completed, add it to ice ether and filter to obtain a black solid product NIR-C6-COOH (53.09 mg, yield 30%) δ 9.05 (d, J = 14.0 Hz, 2H), 8.04–7.85 (m, 6H), 7.66 (t, J = 7.7 Hz, 2H), 7.60 (d, J = 8.3 Hz, 2H), 7.51 (t, J = 7.8 Hz, 2H),7.30(d,J=8.4Hz,2H),7.16(d,J=7.3Hz,2H),6.56(d,J=14.0Hz,2H),4.12(t,J=7.4Hz,4H),2.86(t,J=6.3Hz,4H),2.12(d, J=7.2Hz,2H),1.82(p,J=7.7Hz,4H),1.39(d,J=8.2Hz,4H),1.32–1.25(m,8H),0.84(t,J=6.9Hz,6H).HRMS(ESI)m / z:calculated forC 51 H 53 N2O2S + ,[M] + 757.38223, found 757.38214.;

[0104]

[0105] 11. Dissolve 1.0 mmol of NIR-C6-COOH obtained in step 10, 0.0706 mmol of N,N-diisopropylethylamine (DIPEA) and 0.113 mmol of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) in 4 mL of DMF, and stir at room temperature for 1 h to obtain a reaction solution; dissolve 1.30 mmol of distearoylphosphatidylethanolamine in 1 mL of DMF, add the solution to the reaction solution, and stir at room temperature for 2 h; after the reaction is completed, concentrate in vacuo, and purify by thin layer chromatography (the volume ratio of dichloromethane to methanol is 10:1) to obtain a black solid product NIR-C6-COOH-Lipid (14.53 mg, yield 30%). 1 H NMR (400MHz, CDCl3: MeOD=10:1) δ9.03 (d, J=14.0Hz, 2H), 7.96 (dd, J=17.3, 7.8Hz, 5H), 7.82 (d, J=8.3Hz, 2H), 7.68 (t, J=7.7Hz,2H),7.58(d,J=8.4Hz,2H),7.47(t,J=7.8Hz,2H),7.30(d,J=8.3Hz,2H),7.09(d,J=7.3Hz,2H),6.51(d,J=13. 9Hz,2H),5.09(d,J=6.0Hz,1H),4.31–4.22(m,1H),4.04(d,J=7.6Hz,5H),3.88(t,J=5.6Hz,4H),3.45(d,J=6.2Hz,4H), 2.84(s,4H),2.18(t,J=7.6Hz,4H),1.78(p,J=7.7Hz,5H),1.22–1.15(m,70H),0.81(dt,J=10.8,6.9Hz,15H).MALDITOF m / z calculated for[MI]calculated for C 90 H 129 N3O9PS + 1486.9495,found 1486.762.Abs=1052nm,Em=1100 / 1343nm.

[0106]

[0107] The method for preparing a lipid nanocarrier of a phospholipid molecule C6-Lipid labeled with near-infrared second-zone fluorescence is specifically carried out according to the following steps:

[0108] DMPC, DOPE, DSPE-PEG 2000and C6-Lipid were dissolved in a mixed solution of chloroform and methanol in a volume ratio of 1:1; the mixed solution was evaporated at 30°C in a vacuum environment to form a uniform lipid film; then, the lipid film was dispersed in 1.0mL of phosphate solution and ultrasonicated on an ice bath for 2.5min to obtain a liposome preparation; the liposome preparation was subjected to three heating-cooling cycles between 50°C and 6°C, and then extruded back and forth 10 times through a polycarbonate membrane with a pore size of 400nm and a polycarbonate membrane with a pore size of 200nm using a small extruder, and then the liposomes were dialyzed through a dialysis bag to obtain C6-Lipid-L.

[0109] The method for preparing a lipid nanocarrier of a phospholipid molecule NIR-C6-COOH-Lipid labeled with near-infrared second-zone fluorescence is characterized in that the method is specifically carried out in the following steps:

[0110] DMPC, DOPE, DSPE-PEG 2000 and NIR-C6-COOH-Lipid were dissolved in a mixed solution of chloroform and methanol in a volume ratio of 1:1; the mixed solution was evaporated under a vacuum environment at 30°C to form a uniform lipid film; then, the lipid film was dispersed in 1.0 mL of phosphate solution and ultrasonicated on an ice bath for 2.5 min to obtain a liposome preparation; the liposome preparation was subjected to three heating-cooling cycles between 50°C and 6°C, and then extruded back and forth 10 times through a polycarbonate membrane with a pore size of 400 nm and a polycarbonate membrane with a pore size of 200 nm using a small extruder, and then the liposomes were dialyzed through a dialysis bag to obtain NIR-C6-COOH-Lipid-L.

[0111] Figure 1 Example Compound 2 in CDCl3 1 H NMR spectrum;

[0112] Figure 2 Example C6 in CDCl3 1 H NMR spectrum;

[0113] Figure 3 Example C6-COOH in CDCl3 1 H NMR spectrum;

[0114] Figure 4 Example C6-TPA in CDCl3 1 H NMR spectrum;

[0115] Figure 5 is a high-resolution mass spectrum of Example C6-TPA in CDCl3;

[0116] Figure 6 Example C6-TPE in CDCl31 H NMR spectrum;

[0117] Figure 7 is a high-resolution mass spectrum of Example C6-TPE;

[0118] Figure 8 Example C6-Lipid in CDCl3 1 H NMR spectrum;

[0119] Fig. 9 This is the high-resolution mass spectrum of Example C6-Lipid;

[0120] Fig.10 Example compound 4 in CDCl3 1 H NMR spectrum;

[0121] Fig.11 Example compound 5 in CDCl3 1 H NMR spectrum;

[0122] Fig.12 For example, NIR-C6 in CDCl3 1 H NMR spectrum;

[0123] Fig.13 For example, NIR-C6-COOH in CDCl3 1 H NMR spectrum;

[0124] Fig.14 This is the high-resolution mass spectrum of Example NIR-C6-COOH;

[0125] Fig.15 For example, NIR-C6-COOH-Lipid in CDCl3 1 H NMR spectrum;

[0126] Fig.16 This is the high-resolution mass spectrum of NIR-C6-COOH-Lipid in Example;

[0127] Fig.17 The normalized UV-visible-near infrared absorption spectra of C6-TPA, C6-TPE and C6-Lipid in dichloromethane in Example 1 are shown in the figure. As can be seen from the figure, C6-TPA, C6-TPE and C6-Lipid have strong absorption at 806nm, and C6-TPA and C6-TPE have emission peaks at 908nm. Compared with C6-TPA and C6-TPE, the fluorescence emission of C6-Lipid is slightly red-shifted (915nm).

[0128] Fig.18It is a normalized fluorescence emission spectra of Example C6-TPA, C6-TPE and C6-Lipid in dichloromethane;

[0129] Fig.19 The normalized UV-visible-near infrared absorption spectrum of NIR-C6-COOH-Lipid in dichloromethane in the example is shown in the figure. As can be seen from the figure, the NIR-C6-COOH-Lipid near infrared region II molecule has strong absorption at 1052nm and an emission peak at 1122nm. The above data show that C6-Lipid and NIR-C6-COOH-Lipid have excellent optical properties in the near infrared region.

[0130] Fig. 20 The normalized UV-Vis-NIR absorption spectra of Example C6-TPA-L, C6-TPE-L and C6-Lipid-L are shown in Figure 1. It can be seen that C6-TPA-L, C6-TPE-L and C6-Lipid-L have strong absorption at about 815 nm and strong fluorescence at 923 nm.

[0131] Fig.21 The fluorescence emission spectra of Example C6-TPA-L, C6-TPE-L and C6-Lipid are shown in Figure 1. Compared with C6-TPA-L and C6-TPE-L, C6-Lipid-L has the strongest fluorescence intensity, which is 3.14 times that of C6-TPA-L and 15.7 times that of C6-Lipid-L, and its near-infrared second-zone fluorescence imaging also confirms this.

[0132] Fig. 22 It is the near-infrared second-zone fluorescence imaging image of Example C6-TPA-L, C6-TPE-L and C6-Lipid-L;

[0133] Fig.23 The normalized UV-visible-near infrared absorption spectra of the examples NIR-C6-L and NIR-C6-COOH-Lipid-L; the near-infrared second zone fluorescent molecule NIR-C6-COOH-Lipid also presents a similar pattern. NIR-C6-L has a strong near-infrared absorption peak at 990nm, while NIR-C6-COOH-Lipid-L has a strong absorption peak at 941nm. Compared with NIR-C6-L, NIR-C6-COOH-Lipid-L has a strong emission peak at 1057nm, which is 26.33 times the fluorescence intensity of NIR-C6-L.

[0134] Fig.24 It is the normalized fluorescence emission spectra of Example NIR-C6-L and NIR-C6-COOH-Lipid-L;

[0135] Fig.25 is a transmission electron micrograph of Example C6-Lipid-L;

[0136] Fig.26 is a dynamic light scattering curve diagram of Example C6-Lipid-L;

[0137] Fig. 27 is a transmission electron microscopy image of Example NIR-C6-COOH-Lipid-L;

[0138] Fig.28 This is a dynamic light scattering curve diagram of Example NIR-C6-COOH-Lipid-L.

[0139] Transmission electron microscopy (TEM) and dynamic light scattering images showed that the average hydrodynamic sizes of C6-Lipid-L and NIR-C6-COOH-Lipid-L were approximately 127 and 130 nm, respectively, and the polydispersity index (PDI) was 0.215 and 0.136, respectively, indicating that the nanoparticles were uniform in size. At the same time, the surfaces of C6-Lipid-L and NIR-C6-COOH-Lipid-L were negatively charged (-2.2 mV and -1.23 mV), which was more conducive to their long-term blood circulation and tumor targeting.

[0140] The above tests confirm that the present invention provides a phospholipid molecule labeled with near-infrared second zone fluorescence and a lipid nanocarrier thereof. The use of similar phospholipid-modified organic fluorescent dyes can achieve effective imaging of fluorophores and may produce aggregates such as J aggregation to achieve efficient fluorescence imaging effects.

Claims

1. A method for preparing a phospholipid molecule labeled with near-infrared second region fluorescence, characterized in that The method is specifically carried out in the following steps:

1. Under a nitrogen atmosphere, 1,8-naphtholactimide and iodine hexane were dissolved in toluene, and heated under reflux at 100°C with stirring for 20 hours; after the reaction was completed, the mixture was cooled to room temperature, filtered, and washed with ethyl acetate to obtain compound 2; 2. Add 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde, sodium acetate and compound 2 obtained in step 1 to acetic anhydride, react at 60° C. for 30 min, then cool, pour into saturated sodium bicarbonate solution, wash with brine, and dry to obtain C6; 3. Dissolve mercaptobenzoic acid and C6 obtained in step 2 in a mixed solution of anhydrous dichloromethane and anhydrous methanol, stir at room temperature for 3 hours, and after the reaction is completed, obtain C6-COOH by thin layer chromatography; 4. Dissolve N,N-diisopropylethylamine, HBTU and C6-COOH obtained in step 3 in anhydrous dichloromethane, stir at room temperature for 1 hour to obtain reaction solution 3; dissolve 4-(1,2,2-triphenylvinyl)aniline in anhydrous dichloromethane, add it to reaction solution 3, and stir at room temperature for 24 hours; after the reaction is completed, concentrate in vacuo, and purify by thin layer chromatography to obtain C6-Lipid.

2. The method for preparing a phospholipid molecule labeled with near-infrared second region fluorescence according to claim 1, characterized in that The usage ratio of 1,8-naphtholactimide, iodohexane and toluene in step 1 is 10 mmol: 20 mmol: 20 mL.

3. The method for preparing a phospholipid molecule labeled with near-infrared second region fluorescence according to claim 1, characterized in that In step 2, the usage ratio of acetic anhydride, 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carbaldehyde, sodium acetate and compound 2 is 20 mL: 3.12 mmol: 3.43 mmol: 6.24 mmol.

4. The method for preparing a phospholipid molecule labeled with near-infrared second region fluorescence according to claim 1, characterized in that In step 3, the usage ratio of mercaptobenzoic acid, C6, anhydrous dichloromethane and anhydrous methanol is 0.17mmol:0.083mmol:5mL:5mL.

5. The method for preparing a phospholipid molecule labeled with near-infrared second region fluorescence according to claim 1, characterized in that The usage ratio of N,N-diisopropylethylamine, HBTU, C6-COOH and 4-(1,2,2-triphenylvinyl)aniline in step 4 is 0.070mmol:0.11mmol:0.056mmol:0.084mmol 4-(1,2,2-triphenylvinyl)aniline.

6. The method for preparing a phospholipid molecule labeled with near-infrared second region fluorescence according to claim 1, characterized in that The chemical formula of the C6-Lipid is:

7. A method for preparing a phospholipid molecule labeled with near-infrared second region fluorescence, characterized in that The method is specifically carried out in the following steps: Ⅰ. Under an argon atmosphere, sodium hydride and 1,8-naphtholactimide were dissolved in anhydrous N,N-dimethylformamide; then cooled to 0°C and added to iodohexane; stirred at room temperature for 3 hours. After the reaction was completed, extracted with ethyl acetate, washed with brine and concentrated, purified by silica gel column chromatography, and isolated to obtain compound 4; II. Under argon atmosphere, the compound 4 obtained in step 7 was dissolved in anhydrous THF, methylmagnesium chloride was added, and the mixture was stirred at 55° C. for 1 h. After the reaction was completed, the reaction system was cooled and poured into an ice-water mixture containing HCl solution. THF was removed under reduced pressure, and the aqueous solution of the mixture was filtered. Then, potassium iodide solution was added to obtain a red precipitate. After the solvent was dried under vacuum, potassium iodide solution was added to obtain a red precipitate. The crude product was filtered, and the obtained precipitate was washed three times with ethyl acetate and water, and dried to obtain a solid product 5. III. Add 2-chloro-3-(hydroxymethylene)-cyclohex-1-enecarboxaldehyde and the solid product 5 obtained in step II to 2 mL of acetic acid, and then add to a mixed solution of triethylamine and acetic anhydride, and react at 60° C. for 30 min; after the reaction is completed, cool, add ethyl acetate, filter, and dry to obtain NIR-C6; IV. Dissolve the NIR-C6 obtained in step III and mercaptobenzoic acid in a mixed solution of anhydrous dichloromethane and anhydrous methanol, and stir at room temperature for 2 hours; after the reaction is completed, add to ice ether, filter, and obtain NIR-C6-COOH; V. Dissolve the NIR-C6-COOH, N,N-diisopropylethylamine and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate obtained in step IV in anhydrous dichloromethane, stir at room temperature for 1 hour to obtain a reaction solution; distearoylphosphatidylethanolamine is dissolved in anhydrous dichloromethane, added to the reaction solution, and stirred at room temperature for 2 hours; after the reaction is completed, concentrate in vacuo, and purify by thin layer chromatography to obtain NIR-C6-COOH-Lipid.

8. The method for preparing a phospholipid molecule labeled with near-infrared second region fluorescence according to claim 7, characterized in that The chemical formula of the NIR-C6-COOH-Lipid is:

9. The method for preparing a lipid nanocarrier of a phospholipid molecule labeled with near-infrared second-region fluorescence as claimed in claim 1, characterized in that The method is specifically carried out in the following steps: DMPC, DOPE, DSPE-PEG 2000 and C6-Lipid were dissolved in a mixed solution of chloroform and methanol in a volume ratio of 1:1; the mixed solution was evaporated at 30°C in a vacuum environment to form a uniform lipid film; then, the lipid film was dispersed in 1.0mL of phosphate solution and ultrasonicated on an ice bath for 2.5min to obtain a liposome preparation; the liposome preparation was subjected to three heating-cooling cycles between 50°C and 6°C, and then extruded back and forth 10 times through a polycarbonate membrane with a pore size of 400nm and a polycarbonate membrane with a pore size of 200nm using a small extruder, and then the liposomes were dialyzed through a dialysis bag to obtain C6-Lipid-L.

10. The method for preparing a lipid nanocarrier of phospholipid molecules labeled with near-infrared second-region fluorescence as claimed in claim 7, characterized in that The method is specifically carried out in the following steps: DMPC, DOPE, DSPE-PEG 2000 and NIR-C6-COOH-Lipid were dissolved in a mixed solution of chloroform and methanol in a volume ratio of 1:1; the mixed solution was evaporated under a vacuum environment at 30°C to form a uniform lipid film; then, the lipid film was dispersed in 1.0 mL of phosphate solution and ultrasonicated on an ice bath for 2.5 min to obtain a liposome preparation; the liposome preparation was subjected to three heating-cooling cycles between 50°C and 6°C, and then extruded back and forth 10 times through a polycarbonate membrane with a pore size of 400 nm and a polycarbonate membrane with a pore size of 200 nm using a small extruder, and then the liposomes were dialyzed through a dialysis bag to obtain NIR-C6-COOH-Lipid-L.

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