Preparation method of mitochondrial targeting fluorescent probe and application of mitochondrial targeting fluorescent probe in biological imaging

Abstract

The invention discloses a preparation method of a mitochondrial targeting fluorescent probe and application of biological imaging. The preparation method comprises the following steps: (1) reacting BIS, NIPAM and AAC under the action of an initiator to synthesize nano hydrogel; (2) connecting a ferrous ion fluorescent probe to the nano hydrogel to obtain nano hydrogel with a fluorescent biological probe; and (3) uniformly mixing TPP, DMF, DCC and NHS, adding into the nano hydrogel with the fluorescent biological probe, reacting in a dark place, centrifuging, and washing to obtain the mitochondrial targeting fluorescent probe. The method is mild in synthesis condition, simple and convenient to operate and low in cost, the used chemical reagents have no adverse reaction on human bodies, and industrial production is facilitated; the obtained mitochondrial targeting nano hydrogel shows excellent specificity and sensitivity in Fe < 2 + > detection, and the Fe < 2 + > detection limit is 100 pmol / L.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a preparation method of a mitochondria-targeted fluorescent probe and its application in biological imaging. Background Art

[0002] Abnormal mitochondrial function is closely related to the occurrence and development of many diseases, including metabolic diseases such as iron overload and iron deficiency anemia, as well as neurological diseases such as Parkinson's disease and Alzheimer's disease. 2+ ) plays many important roles within cells.

[0003] First, ferrous ions play a key role in mitochondrial energy production and cell signaling. On the one hand, mitochondria are the cell's energy factories, producing ATP through oxidative phosphorylation, a process that requires the participation of ferrous ions. On the other hand, ferrous ions also participate in intracellular signaling, influencing various physiological and pathological processes. Therefore, measuring the ferrous ion content in mitochondria can help study and evaluate the mechanisms and intervention effects of these diseases.

[0004] However, in monitoring Fe 2+ Conventional fluorescent probes have some limitations when the concentration is low. First, these probes are sensitive to Fe 2+ The responses of Fe ions are easily interfered by other metal ions (such as calcium ions, magnesium ions, and potassium ions), which limits their application in monitoring Fe ions in biological systems. 2+ Accuracy in concentration.

[0005] Furthermore, although high-precision and high-sensitivity detection technologies, such as inductively coupled plasma mass spectrometry (ICP-MS), are widely used, they have high equipment and operating costs and require specialized technicians for operation and maintenance. This limits their use in resource-limited laboratories or for rapid on-site testing.

[0006] In addition, it is also necessary to pay attention to the divalent iron (Fe 2+ ) and ferric iron (Fe 3+ ) may undergo mutual conversion, which may affect their absorption peak positions and peak heights in the atomic absorption spectrum, thereby affecting the accuracy of the detection results.

[0007] Therefore, the development of fluorescent probes with high specificity is crucial to improve the detection accuracy of ferrous ions and other related molecules in biological systems. Summary of the Invention

[0008] The purpose of the present invention is to provide a preparation method of a mitochondrial-targeted fluorescent probe and its application in biological imaging to improve the detection accuracy of ferrous ions in biological systems.

[0009] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is:

[0010] A method for preparing a mitochondrial-targeted fluorescent probe comprises the following steps:

[0011] (1) Under the action of an initiator, N,N'-methylenebisacrylamide, N-isopropylacrylamide and acrylic acid react to synthesize nanohydrogel;

[0012] (2) connecting the ferrous ion fluorescent probe to the nanohydrogel to obtain the nanohydrogel with fluorescent biological probe;

[0013] (3) (3-Carboxypropyl)triphenylphosphonium bromide, N,N-dimethylformamide, N,N′-dicyclohexylcarbodiimide and N-hydroxysuccinimide are mixed and added to the nanohydrogel having the fluorescent biological probe. After the reaction is protected from light, the mixture is centrifuged and washed to obtain the mitochondrial targeting fluorescent probe.

[0014] The preparation method of the present invention has mild synthesis conditions, simple operation, low cost, and the chemical reagents used have no adverse reactions to the human body, which is conducive to industrial production; the obtained mitochondrial targeted nanohydrogel shows excellent specificity and sensitivity in the detection of ferrous ions, and has a high sensitivity to Fe 2+ The detection limit was 100 pmol / L.

[0015] Preferably, step (1) comprises:

[0016] (a) adding N,N'-methylenebisacrylamide, N-isopropylacrylamide, and an emulsifier to deionized water and mixing by ultrasonication;

[0017] (b) continuing to add acrylic acid and initiator, reacting at 55-65° C. for 5.5-6.5 hours under an inert atmosphere, and obtaining the nano-hydrogel after purification.

[0018] Preferably, the molar ratio of N,N'-methylenebisacrylamide to N-isopropylacrylamide is 1:(40-50);

[0019] The initiator includes at least one of ammonium persulfate and V-50 (2,2'-azobisisobutylamidine dihydrochloride);

[0020] The molar ratio of N,N'-methylenebisacrylamide, acrylic acid and ammonium persulfate is 1:5:2.

[0021] Preferably, the purification comprises: first centrifuging the reaction solution at 20,000×g for 60 minutes, taking the supernatant, continuing to centrifuge at 50,000×g for 40 minutes, taking the precipitate, and washing it with 95% ethanol.

[0022] Preferably, step (2) comprises: adding the nano-hydrogel and FeRhoNox-1 aqueous solution to trimethylamine ethanol, stirring overnight at room temperature in the dark; taking the precipitate after centrifugation, and washing it with 95% ethanol.

[0023] Preferably, in step (3), (3-carboxypropyl)triphenylphosphonium bromide, N,N′-dicyclohexylcarbodiimide and N-hydroxysuccinimide are mixed in N,N-dimethylformamide in a molar ratio of 1:4:4;

[0024] The four substances were mixed and stirred at 28-35° C. for 1.5-2.5 h to obtain a mixed solution.

[0025] Preferably, the mass volume ratio of the nano-hydrogel with fluorescent biological probe to the mixed solution is 10 mg:1.0 mL.

[0026] Preferably, in step (3), the nano-hydrogel with the fluorescent biological probe is mixed with the mixed solution, and then stirred for reaction for 10-15 hours under light-proof conditions. After centrifugation, the precipitate is collected and washed with 95% ethanol.

[0027] The present invention also provides a mitochondria-targeted fluorescent probe, and the mitochondria-targeted nanohydrogel is prepared by the above-mentioned preparation method;

[0028] The average particle size of the mitochondria-targeted fluorescent probe is 50-100 nm.

[0029] The hydrogel material of the present invention is in a clear and transparent solution state, and the nanoparticles are dispersed in the solvent. The hydrogel material can be stably stored for more than half a year without solid-liquid separation.

[0030] The present invention also provides application of the mitochondria-targeted fluorescent probe in ferrous ion bioimaging.

[0031] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0032] (1) The preparation method of the present invention has mild synthesis conditions, simple operation, low cost, and the chemical reagents used have no adverse reactions to the human body, which is conducive to industrial production; the obtained mitochondrial targeting nanohydrogel shows excellent specificity and sensitivity in the detection of ferrous ions, and has a high sensitivity to Fe 2+ The detection limit was 100 pmol / L.

[0033] (2) The hydrogel material of the present invention is in a clear and transparent solution state, and the nanoparticles are dispersed in the solvent. It can be stably stored for more than half a year without solid-liquid separation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a transmission electron microscope observation image of the mitochondria-targeted fluorescent probe of the present invention;

[0035] Figure 2 This is a particle size distribution diagram of the mitochondria-targeted fluorescent probe of the present invention;

[0036] Among them, size (d.nm) represents the particle size (diameter, nanometers), Number (percent) represents the number (in percentage);

[0037] Figure 3 This is a fluorescence intensity diagram of the mitochondria-targeted fluorescent probe's ability to detect ferrous ions;

[0038] Wherein, Wavelength represents wavelength, Fluorescence intensity represents fluorescence intensity, the same below;

[0039] Figure 4 This is a statistical diagram of the selection of metal ions by the mitochondria-targeted fluorescent probe of the present invention;

[0040] Figure 5 The mitochondria-targeted fluorescent probe of the present invention is used for biological imaging and its statistical graph;

[0041] Among them, Iron dextran represents iron dextran, yellow channel represents yellow light channel, and green channel represents green light channel;

[0042] Figure 6 This is a comparison chart of the ferrous ion detection capabilities of the mitochondria-targeted fluorescent probe of the present invention and commercial reagents;

[0043] Wherein, Time represents time, mtFe nanosensor represents the mitochondria-targeted fluorescent probe of the present invention, and FeRhoNox-1 is an existing commercial reagent. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0045] Example 1 Preparation of mitochondrial-targeted fluorescent probe

[0046] This embodiment provides a mitochondrial-targeted fluorescent probe, and its preparation method includes the following steps:

[0047] (1) Under the action of an initiator, N,N'-methylenebisacrylamide, N-isopropylacrylamide and acrylic acid react to synthesize nanohydrogel;

[0048] Specifically, the method comprises the following steps: first, adding 2 mmol N,N'-methylenebisacrylamide (BIS), 88 mmol N-isopropylacrylamide (NIPAM) and 0.5 mL Tween 80 to 50 mL deionized water, and mixing by ultrasonic emulsification; then, adding 36 μL acrylic acid (AAC) and 200 μL ammonium persulfate aqueous solution (1 mg / mL), reacting at 60°C for 6 hours under a nitrogen atmosphere, and after completion of the reaction, centrifuging at 20,000 × g for 60 minutes to remove the precipitate, ultracentrifuging the obtained supernatant at 50,000 × g for 40 minutes, taking the precipitate, and washing it three times with 95% ethanol to obtain the nanohydrogel;

[0049] (2) connecting the ferrous ion fluorescent probe to the nanohydrogel to obtain the nanohydrogel with fluorescent biological probe;

[0050] Specifically, the method comprises: taking 1.0 mL (10.0 mg / mL) of the nanohydrogel prepared in step (1) and 100 μL (6.5 μmol / L) of an aqueous solution of FeRhoNox-1, adding 7 μL of trimethylamine ethanol, mixing, and stirring overnight at room temperature in the dark; separating the nanoparticles by centrifugation (50,000 × g, 70 minutes, 4° C.), and washing three times with 95% ethanol to obtain a nanohydrogel with a fluorescent biological probe (hereinafter referred to as NP-2);

[0051] (3) mixing (3-carboxypropyl)triphenylphosphonium bromide, N,N-dimethylformamide, N,N′-dicyclohexylcarbodiimide and N-hydroxysuccinimide, and adding the mixture to the nanohydrogel containing the fluorescent biological probe, centrifuging and washing after the reaction in the dark, thereby obtaining the mitochondrial-targeted fluorescent probe;

[0052] Specifically, the method comprises the following steps: first dissolving 1.695 mg (3-carboxypropyl)triphenylphosphonium bromide (TPP, 3.75 μmol / L) in 1 mL N, N-dimethylformamide (DMF), mixing 15.0 μmol of N, N′-dicyclohexylcarbodiimide (DCC) and 15.0 μmol of N-hydroxysuccinimide (NHS), and stirring at 30° C. for 2 h to obtain a mixed solution; subsequently, adding 100 μL of the mixed solution to 1.0 mL of NP-2 (10.0 mg / mL) and stirring continuously in the dark for 12 h; finally, obtaining the precipitate by ultracentrifugation (50,000×g, 70 minutes), washing three times with 95% ethanol, and then redispersing in 95% ethanol to obtain the mitochondrial-targeted fluorescent probe of this embodiment (concentration of 10.0 mg / mL).

[0053] Take 5 μL of mitochondrial targeted fluorescent probe and drop it onto the ultra-thin carbon film. Observe it under transmission electron microscope. The results are as follows: Figure 1 shown.

[0054] Depend on Figure 1 It can be seen that the mitochondria-targeted fluorescent probe is evenly distributed on the ultrathin carbon film and has good dispersion; particle size analysis (results as shown Figure 2 It was found that the cluster particle size was about 50 nm.

[0055] Example 2 Experiment on the ability of mitochondria-targeted fluorescent probe to detect ferrous ions

[0056] The mitochondria-targeted fluorescent probe prepared in Example 1 was incubated with 1-45 μmol / L ferrous ion solution at a concentration of 200 μg / mL for 30 min, and then its fluorescence intensity was measured. The results are as follows: Figure 3 shown.

[0057] from Figure 3 It can be seen that the mitochondria-targeted nanohydrogel of the present invention has good responsiveness to ferrous ions, and the detection limit of ferrous ions is 100 pmol / L.

[0058] Example 3: Experiment on the selectivity of mitochondria-targeted fluorescent probe for metal ions.

[0059] The mitochondria-targeted fluorescent probe in Example 1 was incubated with different metal ions at a concentration of 200 μg / mL, and its fluorescence intensity was detected. The results were as follows: Figure 4 shown.

[0060] from Figure 4 It can be seen that the mitochondria-targeted nanohydrogel of the present invention has very excellent ion selectivity for ferrous ions, but has no response to other ions.

[0061] Example 4 In vitro cell experiments using mitochondrial-targeted fluorescent probes

[0062] Under physiological conditions, cells produce trace amounts of ferrous ions, which are dynamically balanced in physiological functions. Under abnormal physiological conditions, more ferrous ions are produced, leading to cell death. This example uses human umbilical vein endothelial cells (HUVECs) as the subject to test the ability of the mitochondria-targeted fluorescent probe of Example 1 to detect changes in ferrous ion concentration in cells in vitro.

[0063] HUVEC cells were cultured at 37°C in a 5% CO2 atmosphere. The mitochondria-targeted fluorescent probe (200 μg / mL) was incubated with the cells for 30 min. The cells were then rinsed three times with PBS and observed under a confocal fluorescence microscope. During the observation process, dextran iron was used to intervene in the cells to study the ability to detect intracellular ferrous ions under different physiological states. The results are as follows: Figure 5 shown.

[0064] from Figure 5 It can be seen that with the improvement of modeling drugs, the intracellular ferrous ion content gradually increased, and the mitochondria-targeted fluorescent probe of the present invention can linearly detect this change, indicating that it still shows excellent detection ability in the cellular working environment.

[0065] Example 5 Comparative study of mitochondrial-targeted fluorescent probes and existing commercial reagents

[0066] This example compares the detection capabilities of a commercial probe for detecting intracellular ferrous ions and a mitochondria-targeted fluorescent probe of the present invention (both probes have the same probe loading amount). Specifically:

[0067] HUVEC cells were cultured at 37°C in a 5% CO2 atmosphere, and then the mitochondria-targeted fluorescent probe (200 μg / mL) or FeRhoNox-1 (5 μmol / L) was incubated with the cells for 30 min. The cells were then rinsed three times with PBS and observed under a confocal fluorescence microscope. Figure 6 shown.

[0068] from Figure 6 It can be seen that under the same conditions, the fluorescence lifetime of the mitochondria-targeted fluorescent probe of the present invention is longer than that of the commercial reagent, indicating that the mitochondria-targeted fluorescent probe of the present invention has the ability to dynamically track ferrous ions for a long time.

Claims

1. A method for preparing a mitochondrial targeted fluorescent probe, characterized in that: The following steps are involved: (1) Under the action of an initiator, N,N'-methylenebisacrylamide, N-isopropylacrylamide and acrylic acid are reacted to synthesize a nano-hydrogel; (2) connecting the ferrous ion fluorescent probe to the nanohydrogel to obtain the nanohydrogel with fluorescent biological probe; (3) (3-carboxypropyl)triphenylphosphonium bromide, N,N-dimethylformamide, N,N′-dicyclohexylcarbodiimide and N-hydroxysuccinimide are mixed and added to the nano-hydrogel having the fluorescent biological probe, and the mixture is centrifuged and washed after the reaction in the dark to obtain the mitochondrial targeting fluorescent probe.

2. The preparation method according to claim 1, characterized in that Step (1) comprises: (a) adding N,N'-methylenebisacrylamide, N-isopropylacrylamide and an emulsifier into deionized water and mixing by ultrasonication; (b) continuing to add acrylic acid and initiator, reacting at 55-65° C. for 5.5-6.5 hours under an inert atmosphere, and obtaining the nano-hydrogel after purification.

3. The preparation method according to claim 2, characterized in that: The molar ratio of N,N'-methylenebisacrylamide to N-isopropylacrylamide is 1:(40-50); The initiator includes at least one of ammonium persulfate and 2,2'-azobisisobutylamidine dihydrochloride; The molar ratio of N,N'-methylenebisacrylamide, acrylic acid and ammonium persulfate is 1:5:

2.

4. The preparation method according to claim 2, characterized in that: The purification comprises: first centrifuging the reaction solution at 20,000×g for 60 minutes, taking the supernatant, continuing to centrifuge at 50,000×g for 40 minutes, taking the precipitate, and washing it with 95% ethanol.

5. The preparation method according to claim 1, characterized in that: Step (2) comprises: adding the nano-hydrogel and the FeRhoNox-1 aqueous solution into trimethylamine ethanol, stirring overnight at room temperature in the dark; taking the precipitate after centrifugation, and washing it with 95% ethanol.

6. The preparation method according to claim 5, characterized in that: In step (3), (3-carboxypropyl)triphenylphosphonium bromide, N,N′-dicyclohexylcarbodiimide and N-hydroxysuccinimide are mixed in N,N-dimethylformamide in a molar ratio of 1:4:4; The four substances were mixed and stirred at 28-35° C. for 1.5-2.5 h to obtain a mixed solution.

7. The preparation method according to claim 6, characterized in that: The mass volume ratio of the nano-hydrogel with fluorescent biological probe to the mixed solution is 10 mg:1.0 mL.

8. The preparation method according to claim 7, characterized in that: In step (3), the nano-hydrogel with fluorescent biological probe is mixed with the mixed solution, and then stirred for reaction for 10-15 hours under light-proof conditions. After centrifugation, the precipitate is collected and washed with 95% ethanol.

9. A mitochondrial targeting fluorescent probe, characterized in that: Prepared by the preparation method according to any one of claims 1 to 8; The average particle size of the mitochondria-targeted fluorescent probe is 50-100 nm.

10. Use of the mitochondria-targeted fluorescent probe as claimed in claim 9 in ferrous ion bioimaging.

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