Use of a Class of Rosin-Based AIEgens

Abstract

The present invention discloses the use of a class of rosin-based AIEgens. The structural formula of the rosin-based AIEgens is as follows: The above-mentioned rosin-based AIEgens are used for cell fluorescence imaging. The rosin-based AIEgens of the present invention have extremely high fluorescence efficiency, can significantly improve water solubility through MPEG-PLGA modification, have high safety, and good imaging effect.

Description

Technical Field

[0001] The present invention relates to the use of a class of rosin-based AIEgens, belonging to the field of biochemistry. Background Art

[0002] Biomass-based luminescent materials have the characteristics of natural availability, sustainability, biodegradability, and biocompatibility, and their design and preparation have become a research hotspot. When used as fluorescent probes for biofluorescence imaging, due to their strong hydrophobicity, they are prone to aggregation in organisms, resulting in a sharp decrease in fluorescence intensity, showing the aggregation-caused quenching (ACQ) effect, greatly weakening the fluorescence signal, and limiting their practical application in fluorescence imaging technology. The research group led by Academician Tang Benzhong first proposed the concept of aggregation-induced emission (AIE) of organic fluorescent molecules. The unique properties of aggregation-induced emission luminogens (AIEgens) to aggregate and enhance fluorescence in water can achieve low concentration, large fluorescence signal, and long-term tracking, making them more and more widely used in biological detection such as cell imaging.

[0003] Dehydroabietic acid is the largest and most stable component among the resin acids extracted from natural rosin. In previous studies of this research group, when directly applying dehydroabietic acid arylamine compounds to cell imaging, their application efficiency was reduced due to their ACQ effect and hydrophobicity, and the organic solvents used for auxiliary dissolution, when directly applied to cell imaging, may also pose potential hazards to biological applications (Gao Hong, Chinese Academy of Forestry Sciences, 2013). In previous studies, the highest fluorescence quantum yield of the aggregation-induced emission luminogens (AIEgens) synthesized based on dehydroabietic acid in the solid state was only 8.71% (Xumin Cai, et al., Nat. Commun., (2021) 12:1773; Xumin Cai, et al., Dyes and Pigments., (2022) 204:110454). Currently, there is no report on the research of fluorescent probes with AIE characteristics prepared based on dehydroabietic acid, having a higher quantum yield in the aggregated state and good water solubility, and being applied to cell imaging. Summary of the Invention

[0004] The present invention provides the use of a class of rosin-based AIEgens. The rosin-based AIEgens of the present invention have a relatively high fluorescence quantum yield in the aggregated state, and when prepared into nanoparticles, they have good water solubility and have a strong fluorescence signal when applied to cell fluorescence imaging.

[0005] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0006] The use of a class of rosin-based AIEgens, and the structural formula of the rosin-based AIEgens is:

[0007]

[0008] The above rosin-based AIEgens are used for cell fluorescence imaging.

[0009] The inventors have found through research that the rosin-based AIEgens in the present invention all adopt the m-position connection mode. Compared with the p-position connection mode, a more twisted structure can be obtained, which is not conducive to the π-π stacking between molecules and enhances the luminescence efficiency in the aggregated state.

[0010] To improve water solubility, nanoparticles are prepared based on rosin-based AIEgens for cell fluorescence imaging.

[0011] As one specific implementation scheme, the method for preparing nanoparticles based on rosin-based AIEgens is as follows: Add dehydroabietic acid triarylamine-based AIEgens and MPEG-PLGA to THF, add distilled water, continuously ultrasonic for 3 - 5 min, then stir at room temperature for 8 - 12 h to remove tetrahydrofuran, and purify by dialysis, then filter through a 0.22 μm filter membrane to obtain DTPA-mTPE or 2DTPA-mTPE nanoparticles, and store them in a refrigerator at -2 to 3 °C after freeze-drying.

[0012] To further improve water solubility, the mass ratio of the above rosin-based AIEgens to MPEG-PLGA is 1:(6 - 10). If the amount of MPEG-PLGA is too small, the compound cannot be completely encapsulated. If it is too much, there will be a lot of MPEG-PLGA that does not encapsulate the compound.

[0013] To ensure the dispersion effect of the nanoparticles, the volume of the above distilled water used is 1 - 10 times the volume of THF.

[0014] MPEG-PLGA is methoxypolyethylene glycol-b-poly(lactide-co-glycolide), which is a block polymer prepared by connecting the mPEG and PLGA polymer segments. The inventors have found through research that MPEG-PLGA can be used as a carrier material for rosin-based AIEgens, significantly improving the water solubility of rosin-based AIEgens. THF is tetrahydrofuran. The amount of tetrahydrofuran used is based on the ability to dissolve the materials.

[0015] The above cells are any one of tumor cells A549, 7721, 7901 or Hela.

[0016] Technologies not mentioned in the present invention refer to the prior art.

[0017] Beneficial effects:

[0018] (1) The rosin-based AIEgens DTPA-mTPE and 2DTPA-mTPE of the present invention have obvious AIE characteristics, and DTPA-mTPE has a high fluorescence efficiency of 87.8% in the solid state.

[0019] (2) In the present invention, the two rosin-based AIEgens are prepared into nanoparticles with excellent water solubility. The results of cytotoxicity tests show that the cell survival rate reaches 98%, greatly reducing the risk of using organic solvents in cell imaging, and can be used as a fluorescent probe. Brief Description of the Drawings

[0020] Figure 1 shows the UV absorption and fluorescence emission spectra of the compound in THF:

[0021] (a) Normalized UV absorption spectrum of the compound in THF (1×10 -5 mol / L);

[0022] (b) Normalized fluorescence emission spectrum of the compound in THF (1×10 -6 mol / L);.

[0023] Figure 2 shows the fluorescence emission spectra of DTPA-mTPE (a) and 2DTPA-mTPE (b) in H2O / THF mixed solutions with different water fractions (f w ) (1×10 -6 mol / L);

[0024] Figure 3 is a comparison chart of the relationship between the fluorescence intensity I / I0 of DTPA-mTPE and 2DTPA-mTPE and the water content (I0 is the fluorescence intensity of the compound in the solution with f w =0%, and I is the fluorescence intensity of the compound under the corresponding f w );

[0025] Figure 4 is a comparison chart of the relationship between the fluorescence quantum yield of DTPA-mTPE and 2DTPA-mTPE and the water content;

[0026] Figure 5 is the normalized fluorescence emission spectrum of DTPA-pTPE, 2DTPA-pTPE, DTPA-mTPE, and 2DTPA-mTPE in the solid state;

[0027] Figure 6 is a comparison chart of the fluorescence quantum yields of DTPA-pTPE, 2DTPA-pTPE, DTPA-mTPE, and 2DTPA-mTPE in THF and the solid state;

[0028] Figure 7Synthesis routes of DTPA-mTPE and 2DTPA-mTPE nanoparticles (NPs);

[0029] Figure 8 TEM images of DTPA-mTPE NPs (a) and 2DTPA-mTPE NPs (b);

[0030] Figure 9 Nanoparticle size (DLS) graphs of DTPA-mTPE NPs (a) and 2DTPA-mTPE NPs (b) (2.2 mg / mL);

[0031] Figure 10 UV absorption and fluorescence emission spectra of DTPA-mTPE NPs and 2DTPA-mTPE NPs;

[0032] Figure 11 Cytotoxicity graphs of DTPA-mTPE NPs and 2DTPA-mTPE NPs (In the figure, there are six groups of bar graphs corresponding to six concentrations. In the same group of bar graphs, on the left is 2DTPA-mTPE NPs and on the right is DTPA-mTPE NPs);

[0033] Figure 12 Fluorescence imaging graphs of DTPA-mTPE NPs and 2DTPA-mTPE NPs in A549 lung cancer cells (20 μg / ml, 4 h), where Probes is the confocal imaging graph of only adding nanoparticles in A549 cells, Bright is the confocal imaging graph of only adding the nuclear dye CytoGreen in cells, and Merged is the confocal imaging graph of adding nanoparticles and the nuclear dye CytoGreen in cells. Detailed implementation methods

[0034] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with embodiments. However, the content of the present invention is not limited to the following embodiments only.

[0035] In each example, if the temperature is not specifically stated, the operation is carried out at room temperature (15 - 25 °C).

[0036] Example 1

[0037] The specific synthesis method of DTPA-mTPE is as follows:

[0038] 10 mmol of methyl 12-bromodehydroabietate (a) and 12 mmol of aniline (Aladdin) were added to a three-necked flask, and 45 mL of anhydrous o-xylene was added. The mixture was purged with nitrogen. 14.4 mmol of sodium tert-butoxide, 1.2 mmol of tri-tert-butylphosphine, and 0.3 mmol of palladium acetate were added. The mixture was heated under reflux for 12 h, extracted with ether and water, and the organic phase was collected, dried over anhydrous magnesium sulfate, filtered, rotary evaporated, and purified by column chromatography (V (petroleum ether) : V (ethyl acetate) = 10 : 1) to obtain intermediate b.

[0039] 10 mmol of compound b and 12 mmol of p-bromoanisole were added to a three-necked flask, 45 mL of anhydrous o-xylene was added, and nitrogen was passed through. 14.4 mmol of sodium tert-butoxide, 1.2 mmol of tri-tert-butylphosphine and 0.3 mmol of palladium acetate were added. The mixture was heated under reflux for 12 h, extracted with ether and water, and the organic phase was collected, dried over anhydrous magnesium sulfate, filtered, rotary evaporated, and separated and purified by column chromatography (V (petroleum ether) : V (ethyl acetate) = 25 : 1) to obtain 12-DTPA-OMe.

[0040] Add 10 mmol of the compound 12-DTPA-OMe, 10 mmol of NBS (N-bromosuccinimide), and 250 mL of anhydrous acetonitrile to a round-bottom flask and react in the dark at 25°C for 24 h. Then, rotary evaporation and column chromatography separation and purification (V (petroleum ether) : V (ethyl acetate) = 20 : 1) were performed to obtain 12-DTPA-OMe-Br.

[0041] 1 mmol of the compound 12-DTPA-OMe-Br, 1 mmol of the compound mTPE (Zhengzhou Alpha Chemical Co., Ltd.), 0.025 mmol of Pd(PPh3)4 (tetrakis(triphenylphosphine)palladium), and 0.07 mol of K2CO3 were added to a three-necked flask. 50 mL of THF and 7 mL of H2O were added, and the mixture was refluxed at 70-80°C under a nitrogen atmosphere for 12 hours, then cooled to room temperature. The organic phase was collected, dried over anhydrous magnesium sulfate, and purified by column chromatography (V(petroleum ether) : V(ethyl acetate) = 10 : 1) to obtain DTPA-pTPE, as shown in the following reaction formula:

[0042]

[0043] Example 2

[0044] The specific synthesis method of 2DTPA-mTPE is as follows:

[0045] 10 mmol of methyl 12-bromodehydroabietate (a) and 12 mmol of aniline (Aladdin) were added to a three-necked flask, and 45 mL of anhydrous o-xylene was added. The mixture was purged with nitrogen. 14.4 mmol of sodium tert-butoxide, 1.2 mmol of tri-tert-butylphosphine, and 0.3 mmol of palladium acetate were added. The mixture was heated under reflux for 12 h, extracted with ether and water, and the organic phase was collected, dried over anhydrous magnesium sulfate, filtered, rotary evaporated, and purified by column chromatography (V (petroleum ether) : V (ethyl acetate) = 10 : 1) to obtain intermediate b.

[0046] 10 mmol of compound b and 12 mmol of p-bromoanisole were added to a three-necked flask, 45 mL of anhydrous o-xylene was added, and nitrogen was passed through. 14.4 mmol of sodium tert-butoxide, 1.2 mmol of tri-tert-butylphosphine and 0.3 mmol of palladium acetate were added. The mixture was heated under reflux for 12 h, extracted with ether and water, and the organic phase was collected, dried over anhydrous magnesium sulfate, filtered, rotary evaporated, and separated and purified by column chromatography (V (petroleum ether) : V (ethyl acetate) = 25 : 1) to obtain 12-DTPA-OMe.

[0047] Add 10 mmol of the compound 12-DTPA-OMe, 10 mmol of NBS (N-bromosuccinimide), and 250 mL of anhydrous acetonitrile to a round-bottom flask and react in the dark at 25°C for 24 h. Then, rotary evaporation and column chromatography separation and purification (V (petroleum ether) : V (ethyl acetate) = 20 : 1) were performed to obtain 12-DTPA-OMe-Br.

[0048] 2 mmol of the compound 12-DTPA-OMe-Br, 1 mmol of the compound m,m-TPE (Zhengzhou Alpha Chemical Co., Ltd.), 0.05 mmol of Pd(PPh3)4, and 0.14 mol of K2CO3 were added to a three-necked flask. 50 mL of THF and 7 mL of H2O were added, and the mixture was refluxed at 70-80°C under a nitrogen atmosphere for 12 hours, then cooled to room temperature. The organic phase was collected, dried over anhydrous magnesium sulfate, and purified by column chromatography (V(petroleum ether) : V(dichloromethane) = 5:1) to obtain 2DTPA-mTPE, as shown in the following reaction formula:

[0049]

[0050] As shown in Figure 1, the ultraviolet absorption wavelengths of DTPA-mTPE and 2DTPA-mTPE in THF are 323 and 322 nm, respectively, the fluorescence emission wavelengths are 420 and 432 nm, and the Stokes shifts are 94 and 110 nm, respectively. This indicates that the different numbers of dehydroabietic acid triarylamines affect the fluorescence emission wavelength of the compounds and have Stokes shifts suitable for biological imaging.

[0051] As can be seen from Figures 2, 3, and 4, with the increase in water content, the fluorescence intensity and fluorescence quantum yield of the compound increase, and both DTPA-mTPE and 2DTPA-mTPE exhibit obvious AIE characteristics.

[0052] As Figure 5 can be seen, the fluorescence emission wavelengths of DTPA-mTPE and 2DTPA-mTPE in the solid state are 478 and 475 nm, respectively, indicating that the increase in the dehydroabietic acid skeleton reduces the conjugation degree of the compound, resulting in a blue shift of the emission wavelength.

[0053] As Figure 6 can be seen, DTPA-mTPE has the highest fluorescence quantum yield of 87.8% in the solid state, and the fluorescence quantum efficiency of 2DTPA-mTPE is relatively low at 20.8%. This may be because two dehydroabietic acids are not conducive to the delocalization of electrons, resulting in the dissipation of the energy of the excited state through non-radiative decay. In addition, the fluorescence quantum yields of DTPA-mTPE and 2DTPA-mTPE in the solid state are both higher than those in THF, indicating that the compound has a higher luminescence efficiency in the aggregated state.

[0054] Example 3

[0055] As Figure 7 shown, DTPA-mTPE nanoparticles were prepared by the nanoprecipitation method: A solution of DTPA-mTPE (2 mg) and MPEG-PLGA (Mw: 2000 - 2000, 18 mg) was added to 1 mL of THF, and then 9 mL of distilled water was added. The mixture was continuously sonicated (720 W) for 3 min. The mixture was stirred overnight (12 h) at room temperature to remove tetrahydrofuran, further purified by dialysis, and then filtered through a 0.22 μm filter membrane to obtain DTPA-mTPE nanoparticles (DTPA-mTPE NPs) with a concentration of 2.2 mg / mL.

[0056] 2DTPA-mTPE nanoparticles were prepared by the nanoprecipitation method: 2DTPA-mTPE (2 mg) and MPEG-PLGA (Mw: 2000 - 2000, 18 mg) were added to 1 mL of THF, and then 9 mL of distilled water was added. The mixture was continuously sonicated (720 W) for 3 min. The mixture was stirred overnight (12 h) at room temperature to remove tetrahydrofuran, further purified by dialysis, and then filtered through a 0.22 μm filter membrane to obtain 2DTPA-mTPE nanoparticles (2DTPA-mTPE NPs) with a concentration of 2.2 mg / mL.

[0057] As Figure 8 can be seen, DTPA-mTPE and 2DTPA-mTPE nanoparticles have been synthesized.

[0058] The following nanoparticle tests, unless otherwise specified, were conducted using the 2.2 mg / mL nanoparticle solution prepared above:

[0059] The hydrated diameter and particle size distribution of the nanoparticles before freeze-drying were determined by DLS. Figure 9 As can be seen, the DTPA-mTPE and 2DTPA-mTPE nanoparticles have a particle size of 83.9 nm and 80.4 nm, respectively, and are uniformly distributed in water at a concentration of 2.2 mg / mL. Nanoparticles were prepared according to the aforementioned method using a mass ratio of DTPA-mTPE or 2DTPA-mTPE to MPEG-PLGA of 1:9. The amount of tetrahydrofuran (THF) was determined to dissolve the DTPA-mTPE or 2DTPA-mTPE. After sonication, the THF was removed by stirring at room temperature. The resulting mixture was further purified by dialysis and filtered through a 0.22 μm filter membrane. The concentration of DTPA-mTPE and 2DTPA-mTPE in water reached 50 mg / mL, demonstrating that the modified DTPA-mTPE and 2DTPA-mTPE with MPEG-PLGA exhibits excellent water solubility. For convenient storage and use, the resulting nanoparticles can be freeze-dried and stored in a refrigerator at 3°C for immediate use.

[0060] Depend on Figure 10 It can be seen that the UV absorption wavelengths of DTPA-mTPE and 2DTPA-mTPE nanoparticles are 323 nm and 319 nm, and the fluorescence emission wavelengths are 476 nm and 471 nm, respectively, which is consistent with the f w = 95% similarity in fluorescence emission wavelength.

[0061] Cell proliferation was detected by CCK-8 assay: cells were digested, counted, and prepared to a concentration of 5 × 10 4 Cells (A549 cells, Jiangsu KeyGen Biotech Co., Ltd.) with a suspension of 100 μL / mL were added to each well of a 96-well cell culture plate; the 96-well cell culture plate was placed in a 37°C, 5% CO2 incubator for 24 hours to adhere to the wall; 100 μL of the corresponding culture medium (90% DMEM + 10% FBS) containing 2.5, 5, 10, 20, 40, 80 μg / mL nanoparticles was added according to the group setting, and a negative control group was set up at the same time. The 96-well cell culture plate was placed in a 37°C, 5% CO2 incubator for another 24 hours; the 96-well plate was stained with CCK-8, and the OD value was measured at a wavelength of 450 nm using a microplate reader. Figure 11 It can be seen that the survival rates of A549 lung cancer cells are higher than 95% and even reach 98%, indicating that both DTPA-mTPE and 2DTPA-mTPE nanoparticles have low cytotoxicity.

[0062] Example 4

[0063] Application of Dehydroabietic Acid Triarylamino AIEgens in Cell Imaging:

[0064] Digest the cells in the logarithmic growth phase (Jiangsu Kaygen Biotechnology Co., Ltd., A549 cells) and inoculate them into a confocal dish, with 1×10 4 cells per dish, and wait for the cells to adhere the next day;

[0065] According to the groups, set the cells to incubate with 20 μg / mL DTPA-mTPE and 2DTPA-mTPE nanoparticles for 4 h respectively. At the same time, set up a blank control group and wash it 3 times with PBS;

[0066] Dilute CytoGreen in a ratio of 1:1000 and add it to the confocal dish. After incubating in the dark for 15 min, wash it 3 times with PBS for cell imaging;

[0067] Figure 12 For the cell imaging results of the nanoparticles, as Figure 12 shown, the fluorescence intensities presented by the nanoparticles in A549 lung cancer cells are inconsistent. The intensity in the cytoplasm is higher and weaker in the nucleus, indicating that the compound is enriched in the cytoplasm. The imaging is clear and bright, and it can be used as a cytoplasmic probe; and it can be clearly seen from the figure that the imaging effect of DTPA-mTPE is significantly better than that of 2DTPA-mTPE.

[0068] Comparative Example 1

[0069] DTPA-pTPE and DTPA-mTPE are connected through the p-position and m-position respectively. Comparing the two compounds, it can be seen from Figure 1(a) that the maximum ultraviolet absorption wavelengths of DTPA-pTPE and DTPA-mTPE in THF are 356 and 323 nm respectively; it can be seen from Figure 1(b) that the fluorescence emission wavelengths of DTPA-pTPE and DTPA-mTPE are 433 and 420 nm respectively; by Figure 5 comparison, it can be seen that the fluorescence emission wavelengths of DTPA-pTPE and DTPA-mTPE in the solid state are 488 and 478 nm respectively; this may be because the connection method at the m-position makes the compound have a more twisted configuration, reducing the conjugation degree of the compound and causing the fluorescence emission wavelength to blue-shift. By Figure 6 comparison, it can be seen that the fluorescence quantum yield of DTPA-mTPE is 87.8%, significantly higher than 65.3% of DTPA-pTPE. One of the possible reasons is that the connection method at the m-position makes the compound have a more twisted configuration, which is not conducive to the π-π stacking between molecules in the aggregated state, thus increasing the fluorescence quantum yield of the compound.

[0070] The structure of DTPA-pTPE is as follows:

[0071]

[0072] Comparative Example 2

[0073] 2DTPA-pTPE and 2DTPA-mTPE are connected through the p-position and m-position respectively. By comparing the two compounds, it can be seen from Figure 1(a) that the maximum ultraviolet absorption wavelengths of 2DTPA-pTPE and 2DTPA-mTPE in THF are 360 and 322 nm respectively; it can be seen from Figure 1(b) that the fluorescence emission wavelengths of 2DTPA-pTPE and 2DTPA-mTPE are 433 and 432 nm respectively. This shows that the change in the connection method has little effect on the emission wavelengths of the two compounds with dehydroabietic acid skeletons; Figure 5 It can be seen that the fluorescence emission wavelengths of 2DTPA-pTPE and 2DTPA-mTPE in the solid state are 523 and 475 nm respectively; this may be because the connection method at the m-position makes the compound have a more twisted configuration, reducing the conjugation degree of the compound and causing the fluorescence emission wavelength to blue-shift.

[0074] The structure of 2DTPA-pTPE is as follows:

[0075]

Claims

1. Use of a class of rosin-based AIEgens, characterized by: The structural formula of rosin-based AIEgens is: ； The above-mentioned rosin-based AIEgens were used for cell fluorescence imaging as cytoplasmic probes.

2. Use of the rosin-based AIEgens as described in claim 1, characterized in that: Nanoparticles were prepared based on rosin-based AIEgens for cell fluorescence imaging.

3. Use of the rosin-based AIEgens according to claim 2, characterized in that: The method for preparing nanoparticles based on rosin-based AIEgens is as follows: rosin-based AIEgens and MPEG-PLGA are added to THF, distilled water is added, and continuous ultrasound is performed for 3 to 5 minutes, followed by stirring at room temperature for 8 to 12 hours. The nanoparticles are purified by dialysis and then filtered through a 0.22 μm filter membrane to obtain DTPA-mTPE nanoparticles. After freeze-drying, the nanoparticles are stored in a refrigerator at -2 to 3°C. MPEG-PLGA is methoxypolyethylene glycol-b-polylactide-glycolide.

4. Use of the rosin-based AIEgens according to claim 3, characterized in that: The mass ratio of rosin-based AIEgens and MPEG-PLGA was 1:(6~10).

5. Use of the rosin-based AIEgens according to claim 3, characterized in that: The volume of distilled water used is 1 to 10 times the volume of THF.

6. Use of the rosin-based AIEgens according to any one of claims 1-5, characterized in that, The cells are any one of tumor cells A549, 7721, 7901 or Hela.

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