Water-soluble fluorescent probe and nanoparticals with aggregation-induced emission effect for ovarian cancer and preparation method and use thereof

Abstract

Disclosed are a water-soluble fluorescent probe and nanoparticles with an aggregation induced emission (AIE) effect and preparation methods and the application. The fluorescent probe molecule includes one imidazole ring substituted by two methoxyphenyls and one phenyl and two quaternary ammonium salt structures. It has good dispersion in water, may form micelles. It has the characteristics of AIE effect and a large Stokes shift etc. It is suitable for fluorescence detection. Under the induction of lysophosphatidic acid, the probe can be self-assembled so as to aggregate and generate yellow fluorescence. Lysophosphatidic acid may be quantitatively analyzed by measuring the intensity of emitted fluorescence.

Claims

1. A fluorescent molecular probe, wherein the fluorescent molecular probe has a structure of Formula 1: ##STR00005## wherein the fluorescent molecular probe is water-soluble and has an aggregation-induced emission effect, and the fluorescent molecular probe is used for specifically recognizing lysophosphatidic acid.

2. The fluorescent molecular probe of claim 1, wherein the fluorescent molecular probe is formed by linking a tetra-substituted imidazole ring, a dodecyl chain and a pyridine ring with formation of a salt.

3. A fluorescent nanoprobe, wherein the fluorescent nanoprobe is formed by self-assembly of the fluorescent molecular probe of claim 1, and the fluorescent nanoprobe is used for specifically recognizing the lysophosphatidic acid.

4. A method for preparing the fluorescent molecular probe of claim 1, wherein the method comprises the following steps: 1) 4-formylpyridine, aniline and anisil undergoing a one-pot cyclization reaction in an acetic acid/ammonium acetate system, to obtain an intermediate of Formula 2; 2) the intermediate of Formula 2 and 1,12-dibromododecane undergoing a nucleophilic substitution reaction, to obtain an intermediate of Formula 3; and 3) the intermediate of Formula 3 and pyridine undergoing a nucleophilic substitution reaction, to obtain a target product, ##STR00006##

5. The method of claim 4, wherein: a reaction process in the step 1) is as follows: dissolving the 4-formylpyridine and the aniline in a glacial acetic acid solvent and stirring for 0.5-1.5 hours at room temperature, and then adding the anisil and ammonium acetate, reacting for 6-12 hours at 120 C.; reaction conditions in the step 2) are as follows: using acetonitrile as a solvent, and reacting for 6-10 hours at 90 C.; and reaction conditions in the step 3) are as follows: using the pyridine as a solvent, reacting for 6-10 hours at 90 C.

6. A method for preparing the fluorescent nanoprobe of claim 3, comprising: dissolving the fluorescent molecular probe into an organic solvent, to yield an obtained mixture, adding the obtained mixture into an aqueous solution, performing ultrasonic treatment, and obtaining the fluorescent nanoprobe.

7. The method of claim 6, wherein the organic solvent is selected from at least one of methanol, ethanol, dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, acetone, or acetonitrile, and the aqueous solution is selected from pure water, physiological saline, a phosphate buffer solution (PBS), a tris(hydroxymethyl)aminomethane hydrochloride buffer solution or a 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a dynamic light scattering diagram of the fluorescent molecular probe and nanoparticles prepared according to the disclosure.

(2) FIG. 2 is a scanning electron micrograph showing recognition of lysophosphatidic acid by the fluorescent molecular probe and nanoparticles according to the disclosure.

(3) FIG. 3 shows the linear relationship between the fluorescence intensity of the fluorescent molecular probe and nanoparticles according to the disclosure and the concentration of lysophosphatidic acid. An abscissa represents the concentration of lysophosphatidic acid, and an ordinate represents the fluorescence intensity.

(4) FIG. 4 shows the selectivity of the fluorescent molecular probe and nanoparticles according to the disclosure to lysophosphatidic acid.

(5) FIG. 5 is a schematic diagram showing the mechanism for detecting lysophosphatidic acid by the fluorescent molecular probe and nanoparticles.

DETAILED DESCRIPTION

(6) The following Examples are intended to further illustrate the disclosure.

Example 1

(7) Compound 3 with enhanced aggregation-induced fluorescence emission of the disclosure was synthesized with the followed synthetic route.

(8) ##STR00004##
Synthesis of Compound 1

(9) Aniline (93.1 mg, 1 mmol) and 4-formylpyridine (107.1 mg, 1 mmol) were weighed respectively and dissolved in 6-8 mL of glacial acetic acid. The obtained mixture was stirred for 1 hour at room temperature. Anisil (207.2 mg, 1 mmol) and ammonium acetate (462.5 mg, 6 mmol) were added in sequence into the reaction system. The reaction lasted overnight at 120 C., and the reaction was quenched with water. The reaction system was poured into 200 mL of iced water. A pH of the system was adjusted to neutral by using 0.1 mmol/L of sodium hydroxide solution. The mixture was filtered and washed with water for three times. After being dried under vacuum, a product was obtained by purifying with the silica gel column chromatography in a yield of 21.9%. Results of nuclear magnetic resonance analysis were as follows: .sup.1H NMR (500 MHz, CDCl.sub.3) 8.47 (d, 2H), 7.55 (d, 2H), 7.45-7.25 (m, 5H), 7.09 (t, 2H), 7.05 (d, 2H), 6.83 (d, 2H), 6.78 (d, 2H), 3.79 (d, 6H). .sup.13C NMR (100 MHz, CDCl.sub.3) 159.40, 158.64, 149.59, 143.36, 136.80, 132.29, 126.77, 122.18, 113.95, 113.71, 55.20, 55.13.

(10) Synthesis of Compound 2

(11) The compound 1 (191.5 mg, 0.44 mmol) and 1,12-dibromododecane (145.0 mg, 0.44 mmol) were weighed and dissolved in 3 mL of acetonitrile solution, and fully stirred at room temperature. The reaction system was fully refluxed for 8 hours at 90 C. After the reaction was completed by monitoring with TLC plates, the solvents were removed by distilling under vacuum. The compound 2 was purified by the silica gel column chromatography in a yield of 34.7%. Results of nuclear magnetic resonance analysis were as follows: .sup.1H NMR (500 MHz, DMSO-d6) 8.88 (d, 2H), 7.74 (d, 2H), 7.49 (dd, 7H), 7.20 (d, 2H), 6.90 (d, 4H), 4.45 (t, 2H), 3.73 (d, 6H), 3.51 (t, 2H), 1.79 (dd, 4H), 1.23 (s, 16H). .sup.13C NMR (125 MHz, DMSO-d6) 159.99, 159.14, 132.75, 130.42, 130.10, 128.97, 128.24, 126.12, 123.70, 121.22, 114.57, 114.37, 60.38, 55.57, 35.67, 32.68, 30.77, 29.31, 29.28, 29.18, 28.78, 28.55, 27.95, 25.82.

(12) Synthesis of Compound 3

(13) The compound 2 (155.0 mg, 0.20 mmol) was weighed and dissolved in 4-5 mL of pyridine solution, and fully stirred at room temperature. The reaction system was fully refluxed for 8 hours at 90 C. After the reaction was completed by monitoring with TLC plates, the solvents were removed by distilling under vacuum. The target compound 3 was produced by the silica gel column chromatography in a yield of 68.2%. Results of nuclear magnetic resonance analysis were as follows: .sup.1H NMR (500 MHz, CD.sub.3OD) 9.11-8.98 (m, 2H), 8.85-8.66 (m, 2H), 8.60 (t, 1H), 8.12 (s, 2H), 7.86 (d, 2H), 7.50 (d, 5H), 7.38 (s, 2H), 7.13 (d, 2H), 6.84 (s, 4H), 4.66 (s, 2H), 4.50 (s, 2H), 3.89-3.62 (m, 6H), 1.99 (d, 4H), 1.56-1.04 (m, 16H). .sup.13C NMR (125 MHz, CD.sub.3OD) 160.33, 159.48, 145.45, 144.54, 144.41, 143.81, 143.77, 141.37, 139.62, 139.58, 136.00, 132.17, 132.14, 129.91, 129.88, 128.32, 128.10, 123.66, 120.69, 113.79, 113.76, 113.41, 113.39, 61.72, 60.69, 54.38, 54.33, 31.10, 30.84, 30.81, 29.76, 29.72, 29.36, 29.33, 29.13, 29.05, 28.68, 25.77.

Example 2

(14) Preparation of the Water-Soluble Organic Fluorescent Molecular Probe

(15) 40 L of compound 3 (1 mM) in a phosphate buffer solution was taken by using a pipette and placed in an ep-tube. 1960 L of a phosphate buffer solution (pH=7.4) was added into the ep-tube under an ultrasonic condition so that the final concentration of compound 3 was 20 M. The mixture was stirred for 30 min under room temperature to generate opalescence. In order to verify nano-aggregation behavior thereof, an average particle size thereof measured by a dynamic light scattering experiment was 600 nm, as shown in FIG. 1.

Example 3

(16) Testing Recognition of Lysophosphatidic Acid by the Prepared Fluorescent Molecular Probe and Nanoparticles Through a Scanning Electron Microscope (SEM)

(17) 40 L of compound 3 (1 mM) in a phosphate buffer solution was taken by using a pipette and placed in an ep-tube. 1900 L of a phosphate buffer solution (pH=7.4) was added under an ultrasonic condition, and 60 L of lysophosphatidic acid (1 mM) in a phosphate buffer solution was added so that the final concentration of the compound 3 in the solution was 20 M, and the final concentration of lysophosphatidic acid was 30 M. The mixture was stirred for 30 min at room temperature. A drop of the prepared solution was sucked, and dropped onto a copper grid. Water was absorbed by filter paper. Then the copper grid was air-dried and placed in a transmission electron microscope for observation. A transmission electron micrograph is shown in FIG. 2.

Example 4

(18) A Linear Relationship Between the Fluorescence Intensity of the Fluorescent Molecular Probe and Nanoparticles and the Concentration of Lysophosphatidic Acid

(19) A series of different volumes of the stock solution of lysophosphatidic acid in the phosphate buffer solution were respectively added to the system prepared in Example 2, so that the final concentrations of lysophosphatidic acid respectively were 0 M, 0.2 M, 0.4 M, 0.8 M, 1.0 M, 1.2 M, 1.4 M, 1.8 M, 2.0 M, 4.0 M, 8.0 M, 10.0 M, 12.0 M, 14.0 M, 16.0 M, 18.0 M, 20.0 M, 25.0 M, 30.0 M, 33.0 M. After all of testing solution were prepared, the testing solutions were mixed uniformly with a vortex. After being incubated for 1 min at room temperature, the fluorescence emission intensity thereof at 557 was measured. As shown in FIG. 3, the results show there is a good linear relationship between the fluorescence intensity of the system at 557 nm and the concentration of lysophosphatidic acid in the range of 0-33 M.

Example 5

(20) Selectivity of the Organic Fluorescent Nanoprobe to Detection of Lysophosphatidic Acid

(21) The prepared fluorescent molecular probe solution was used for evaluating the selectivity of the probe to lysophosphatidic acid. An excitation wavelength of the nanoprobe was 400 nm. The concentration of the compound 3 in the system was 10 M. Lysophosphatidic acid with a final concentration of 50 M and L-proline, glycerin, sodium phosphate, magnesium sulfate, sodium acetate, sodium nitrate, sodium fluoride, glucose, urea, and lysophosphatidyl choline (LPC) with a final concentration of 1 mM were respectively added into the system. After fully mixing, the systems were incubated for 1 min at room temperature. Then fluorescence emission spectra thereof were measured and the fluorescence emission intensities at 557 nm were recorded. As shown in FIG. 4, only the system with the addition of lysophosphatidic acid generated strong yellow fluorescence, and the other solutions with the addition of other substances could not be observed to generate yellow fluorescence. The above results show that the organic fluorescent molecular probe and nanoparticles have good selectivity and practical applicability for detecting lysophosphatidic acid.