An organic compound biological probe, and a preparation method and application thereof

Abstract

The application discloses an organic compound biological probe and a preparation method and application thereof, and belongs to the field of fluorescent probes. The structural formula of the organic compound biological probe is as follows, and the preparation method comprises the following steps: adding 2,4-dihydroxybenzaldehyde and anhydrous potassium carbonate into anhydrous acetonitrile or DMF, then dropwise adding 1,4-dibromobutane to obtain a colorless oily compound 1 through reflux reaction; dissolving the compound 1 and triphenylphosphine in anhydrous acetonitrile to obtain a solid compound 2 through reflux reaction; dissolving 1,1,2-trimethyl-1H-benzo[e]indole and iodoethane in anhydrous acetonitrile to obtain a compound 3 through reflux; dissolving the compound 3 and the compound 2 in anhydrous ethanol, adding piperidine, and then performing reflux reaction and purification to obtain the organic compound biological probe. The organic compound biological probe can self-assemble into nanoparticles with a size of about 1.6 nm in water, has strong anti-photobleaching ability, and integrates red light and near-infrared dual-channel imaging, pH detection, mitochondrion targeting and chemotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of fluorescent probes, and in particular relates to an organic compound biological probe and a preparation method and application thereof. Background Art

[0002] Mitochondria participate in processes such as cellular energy supply, cell differentiation, cell signaling, and apoptosis, and possess the ability to regulate cell growth and the cell cycle. The mitochondrial membrane has a negative charge distribution on the inside and a positive charge distribution on the outside. The lipophilic cation triphenylphosphine readily enters the mitochondria along the potential gradient, playing an increasingly important role in the design and synthesis of mitochondrial-targeted drugs. Cancer poses a significant threat to human survival and development, and therefore cancer treatment has received considerable attention. Targeting subcellular organelles can help improve cancer treatment, and mitochondrial-targeted drugs hold great potential for application in cancer treatment.

[0003] Among organic fluorescent probes with different fluorescence emission ranges, those with near-infrared (NIR) fluorescence emission represent greater biological application value, including deeper tissue penetration, lower biological background interference, and less biological damage. Cyanocyanine-derived dyes are easy to functionally design and synthesize, making them ideal NIR fluorophores for the preparation of biodiagnostic and therapeutic agents.

[0004] Intracellular pH is an important physiological determinant of enzyme activity and cellular function. All proteins rely on strictly regulated pH to maintain their structure and function. pH-sensitive biological probes can be used to monitor changes in intracellular pH.

[0005] However, few existing biological probes have the combined properties of pH sensitivity, in vivo and in vitro near-infrared imaging, and chemotherapy capabilities. Summary of the Invention

[0006] The object of the present invention is to provide an organic compound biological probe and its preparation method and application. The organic compound biological probe of the present invention has pH sensitivity, in vivo and in vitro near-infrared imaging and chemotherapy capabilities.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] An organic compound biological probe named Cy-Mito, with the structural formula shown below:

[0009]

[0010] The method for preparing the organic compound biological probe Cy-Mito comprises the following steps:

[0011] 2,4-Dihydroxybenzaldehyde and anhydrous potassium carbonate were added to anhydrous acetonitrile or N,N-dimethylformamide (DMF) and heated with stirring. 1,4-Dibromobutane was added dropwise to carry out reflux reaction. After cooling, solid-liquid separation was performed and the liquid was purified to obtain a colorless oily compound 1.

[0012] The compound 1 and triphenylphosphine were dissolved in anhydrous acetonitrile and refluxed, cooled, and then purified to obtain a solid compound 2.

[0013] 1,1,2-Trimethyl-1H-benz[e]indole and iodoethane were dissolved in anhydrous acetonitrile to obtain a mixture, and the mixture was refluxed. After cooling, the mixture was separated into solid and liquid, and the solid was purified to obtain compound 3.

[0014] The compound 3 and the compound 2 are dissolved in anhydrous ethanol, piperidine is added and reflux reaction is carried out, and the mixture is cooled and purified to obtain solid Cy-Mito, namely the organic compound biological probe.

[0015] The molar ratio of the 2,4-dihydroxybenzaldehyde to the 1,4-dibromobutane is preferably 1:(0.5-1).

[0016] The molar ratio of the compound 1 to the triphenylphosphine is preferably 1:(1-1.5).

[0017] The molar ratio of the 1,1,2-trimethyl-1H-benz[e]indole to the ethyl iodide is preferably 1:(1-5).

[0018] The molar ratio of the compound 2 to the compound 3 is preferably 1:(1-1.5).

[0019] The organic compound biological probe Cy-Mito can self-assemble into nanoparticles in water. Cy-Mito can quickly enter cells within 1 minute, has strong resistance to photobleaching, and integrates dual-channel imaging, pH detection, mitochondrial targeting, and chemotherapy.

[0020] The organic compound biological probe Cy-Mito has the following applications: application in in vivo and in vitro fluorescence imaging, application in mitochondrial targeting, application in pH detection, and application in the preparation of cancer therapeutic drugs.

[0021] The present invention has the following advantages and excellent effects:

[0022] (1) The bioprobe of the present invention is simple to synthesize, which is conducive to commercial promotion and application.

[0023] (2) The characterization and application of the present invention show that Cy-Mito has excellent application prospects, with aggregation-induced luminescence properties, pH sensitivity, dual-channel imaging, mitochondrial targeting, strong resistance to photobleaching and chemotherapy capabilities.

[0024] (3) The Cy-Mito bioprobe of the present invention can achieve near-infrared fluorescence imaging in vivo and in vitro, which has very good application prospects for image-guided chemotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the synthesis process of Cy-Mito.

[0026] Figure 2 This is the H NMR spectrum of Cy-Mito.

[0027] Figure 3 This is the mass spectrum of Cy-Mito.

[0028] Figure 4 It is the normalized UV absorption and solid fluorescence spectrum of Cy-Mito.

[0029] Figure 5 This is a transmission electron micrograph of Cy-Mito.

[0030] Figure 6 Figure 3 is the UV absorption graph of Cy-Mito in Britton-Robinson buffer solutions at different pH values.

[0031] Figure 7 Figure 3 is the fluorescence spectra of Cy-Mito in BF buffer solutions with different pH values.

[0032] Figure 8 This is the MTT result of Cy-Mito in different cell lines.

[0033] Figure 9 It is the intracellular mitochondrial localization of Cy-Mito.

[0034] Figure 10 This is the intracellular dual-channel imaging of Cy-Mito.

[0035] Figure 11 This is the Cy-Mito anti-photobleaching experiment.

[0036] Figure 12 This is the near-infrared fluorescence imaging of Cy-Mito in 4T1 tumor-bearing mice. DETAILED DESCRIPTION

[0037] The technical solutions of the present invention are further described in detail below through specific examples. It should be understood that these examples are only some preferred technical solutions, and the scope of protection requested by the claims is not limited to the following examples.

[0038] Example 1: Organic Synthesis of Cy-Mito ( Figure 1 )

[0039] (1) 1 mmol of 2,4-dihydroxybenzaldehyde and 1.1 mmol of anhydrous potassium carbonate were added to 15 mL of anhydrous acetonitrile and heated with stirring. 1 mmol of 1,4-dibromobutane was added dropwise and the mixture was refluxed for 5 h. After cooling to room temperature, the filtrate was filtered and the solvent was removed by rotary evaporation. Compound 1 was separated and purified using a silica gel column to obtain a colorless oil with a yield of 67%.

[0040] (2) 1 mmol of compound 1 was dissolved in 15 mL of acetonitrile, and 1 mmol of triphenylphosphine was added at room temperature. After reflux reaction for 24 h, the temperature was lowered and rotary evaporation was performed. Compound 2 was obtained by purification using a silica gel column with a yield of 87%.

[0041] (3) 1 mmol of 1,1,2-trimethyl-1H-benz[e]indole and 2 mmol of iodoethane were dissolved in 10 mL of anhydrous acetonitrile. The mixture was refluxed for 12 h. After cooling to room temperature, the solid was filtered and purified using a silica gel column to obtain compound 3 in 85% yield.

[0042] (4) 1 mmol of compound 2 and 1 mmol of compound 3 were dissolved in 20 mL of anhydrous ethanol, 3 drops of piperidine were added, and the mixture was refluxed for 24 h. After the system was cooled to room temperature, the solvent was evaporated under reduced pressure and purified on a silica gel column to obtain solid Cy-Mito with a yield of 89%. The H NMR spectrum is shown in Figure 2 , mass spectrum see Figure 3 , demonstrating the successful synthesis of Cy-Mito.

[0043] Example 2: Characterization of Cy-Mito

[0044] 1. Take 10 μM Cy-Mito in Example 1 and perform UV spectrum test and solid fluorescence test. The results are as follows: Figure 4 As shown. Figure 4 It can be seen that Cy-Mito has a wide visible absorption range and NIR solid luminescence ability. 10μM Cy-Mito was prepared in water, and after ultrasonication for 5 minutes, the sample was prepared and the transmission electron microscope image was taken. The results are as follows Figure 5 shown by Figure 5 It can be seen that Cy-Mito can self-assemble in water to form nanoparticles of about 1.6 nm;

[0045] 2. Take 2 mL of Britton-Robinson buffer solution of different pH values ​​and prepare 10 μM solution with Cy-Mito in Example 1. Detect the ultraviolet absorption of Cy-Mito probe in buffer solution of different pH values. The results are as follows: Figure 6 As shown. Figure 6 It can be seen that within the physiological pH range, as the pH increases, the ultraviolet absorption of Cy-Mito at 464nm gradually decreases, and the ultraviolet absorption at 546nm gradually increases.

[0046] The fluorescence changes of Cy-Mito probe in different pH were detected. Figure 7 shown by Figure 7 It can be seen that within the physiological pH range, as the pH increases, the fluorescence emission peak of Cy-Mito red-shifts from 584 nm to 616 nm, and the fluorescence intensity does not change significantly.

[0047] Example 3: Cytotoxicity of Cy-Mito

[0048] COS-7 healthy cells and three cancer cells (HeLa cells, 4T1 cells and MCF-7 cells) were used as research subjects. Different concentrations of Cy-Mito in Example 1 were incubated with the cells for 24 h, and the cytotoxicity MTT test was performed. The MTT results are shown in Figure 2. Figure 8 As shown, as the concentration of the probe Cy-Mito increases, the survival rate of cancer cells gradually decreases, and the probe has a stronger ability to inhibit the growth of cancer cells, and has the ability to be used in chemotherapy.

[0049] Example 4: Cy-Mito Cell Imaging Test

[0050] After 1 μM Cy-Mito in Example 1 was incubated with HeLa cells for 2 h, commercial mitochondrial localization dye MitoGreen was added and incubated for 15 min. Fluorescence confocal imaging test was performed. The results are shown in Figure 9 The correlation coefficients of mitochondrial localization were all around 0.95, indicating that Cy-Mito has good mitochondrial targeting ability. 5 μM Cy-Mito in Example 1 was incubated with HeLa cells for 2 hours and then fluorescence confocal imaging was performed. The results are shown in Figure 10 , fluorescence signals can be detected in both red and near-infrared fluorescence channels, indicating that Cy-Mito has red and near-infrared dual-channel imaging capabilities. 300 consecutive laser scans were performed, and fluorescence confocal images and signal intensity changes of different imaging channels were collected and analyzed. The results are as follows Figure 11 As shown in the figure, after the scan, the intensity of the red and near-infrared fluorescence channels can still be maintained above 80%, proving that Cy-Mito has excellent anti-photobleaching ability.

[0051] Example 5: In vivo near-infrared imaging of Cy-Mito tumor-bearing mice

[0052] In vivo imaging tests were performed using 4T1 tumor-bearing mice as an animal model. 5 mg / kg of Cy-Mito from Example 1 was injected intratumorally. Near-infrared imaging tests were performed at different time points using a small animal in vivo imaging system. Under 561 nm excitation light, near-infrared fluorescence signals at 710 nm were collected. The results are shown in Figure 2. Figure 12 As shown in the figure, the near-infrared fluorescence intensity in mice was the strongest 0.5 h after the injection of Cy-Mito. As time went on, the near-infrared fluorescence signal gradually disappeared, proving that Cy-Mito has the ability of in vivo near-infrared imaging.

Claims

1. An organic compound bioprobe, characterized in that: Its structural formula is shown below: 。 2. The method for preparing the organic compound bioprobe according to claim 1, characterized in that: The steps include: 2,4-Dihydroxybenzaldehyde and anhydrous potassium carbonate were added to anhydrous acetonitrile or N,N-dimethylformamide and heated under stirring, 1,4-dibromobutane was added dropwise to carry out reflux reaction, and after cooling, solid-liquid separation was performed and the liquid was purified to obtain a colorless oily compound 1; The compound 1 and triphenylphosphine are dissolved in anhydrous acetonitrile and refluxed, cooled, and then purified to obtain a solid compound 2; 1,1,2-trimethyl-1H-benzo[e]indole and iodoethane were dissolved in anhydrous acetonitrile to obtain a mixture, the mixture was refluxed, cooled, solid-liquid separation was performed, and the solid was purified to obtain compound 3; The compound 3 and the compound 2 are dissolved in anhydrous ethanol, piperidine is added and refluxed, and the solid obtained after cooling and purification is the organic compound biological probe; The structural formulas of Compound 1, Compound 2, and Compound 3 are: 、 、 .

3. The method for preparing an organic compound bioprobe according to claim 2, wherein: The molar ratio of the 2,4-dihydroxybenzaldehyde to the 1,4-dibromobutane is 1:(0.5-1).

4. The method for preparing an organic compound bioprobe according to claim 2, wherein: The molar ratio of the compound 1 to the triphenylphosphine is 1:(1-1.5).

5. The method for preparing an organic compound bioprobe according to claim 2, wherein: The molar ratio of the 1,1,2-trimethyl-1H-benz[e]indole to the ethyl iodide is 1:(1-5).

6. The method for preparing an organic compound bioprobe according to claim 2, wherein: The molar ratio of the compound 2 to the compound 3 is 1:(1-1.5).

7. Use of the organic compound bioprobe according to claim 1 in the preparation of cancer therapeutic drugs, characterized in that: The cancer is cervical cancer or breast cancer.

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