A mitochondrial-targeted fluorescent carbon dot and its preparation method and application

Abstract

The present invention relates to the technical field of bio-nanomaterials, and discloses a mitochondrial-targeted fluorescent carbon dot and a preparation method and application thereof. The mitochondrial-targeted fluorescent carbon dot is prepared from a raw material containing p-aminophenol and an organic solvent. In addition, the preparation method of the mitochondrial-targeted fluorescent carbon dot comprises: (1) dissolving p-aminophenol in an organic solvent, and then adjusting the pH value of the obtained mixed solution to 3-5 and then performing a solvothermal reaction to obtain a mitochondrial-targeted fluorescent carbon dot primary product; (2) separating and purifying the mitochondrial-targeted fluorescent carbon dot primary product to obtain a mitochondrial-targeted fluorescent carbon dot aqueous solution. The preparation method is simple to operate and low in cost. The prepared mitochondrial-targeted fluorescent carbon dot exhibits highly selective mitochondrial targeting through hydrophobic interaction and hydrogen bonding rather than traditional electrostatic interaction, and can be used for non-destructive visualization of mitochondria.

Description

Technical Field

[0001] The present invention relates to the technical field of biological nanomaterials, and in particular to mitochondria-targeted fluorescent carbon dots and a preparation method and application thereof. Background Art

[0002] At present, many types of fluorescent substances, such as fluorescent small molecules, inorganic / organic nanoparticles, polymers, and precious metal nanoclusters, have been used for organelle imaging. For mitochondrial localization, the most common method is based on electrostatic adsorption. This is because mitochondria have a special membrane potential (MMP), and the MMP, which is negative inside and positive outside, is more likely to adsorb cations / positive charges. By modifying some organic molecules or nanomaterials with cations / positive charges, commonly used targeting groups include organic phosphine / sulfur salts, quaternary ammonium salts, transition metal complexes, guanidine salts or biguanidine salts. The modified probes can bind well to mitochondria. However, these ideas for functionalization to obtain targeting capabilities are feasible, but on the one hand, they cause complex synthesis and modification steps, and on the other hand, they may cause additional damage to organelles.

[0003] Patent application CN108504349A discloses a method for preparing rhodamine hybrid carbon dots and their application in mitochondrial targeting recognition, the method comprising: dissolving citric acid and m-aminophenol in distilled water, treating at 160°C for 2h by microwave-assisted hydrothermal method, then filtering and drying to obtain rhodamine hybrid carbon dots. The carbon dots rely on traditional electrostatic action to target mitochondria and have a positive charge.

[0004] Patent application CN106833629A discloses a mitochondrial-targeted fluorescent carbon dot and its preparation method and application, the method comprising: dissolving chitosan, ethylenediamine and mercaptosuccinic acid in a dilute hydrochloric acid solution, and reacting at 180°C for 24 hours to obtain mitochondrial-targeted carbon dots by a hydrothermal method. The carbon dots rely on traditional electrostatic effects to target mitochondria, have significant positive charges, and are hydrophilic. Summary of the invention

[0005] The purpose of the present invention is to overcome the problems of complex synthesis and modification steps and possible damage to organelles in common mitochondrial localization methods in the prior art, and to provide a mitochondrial targeted fluorescent carbon dot and a preparation method thereof, as well as the use of the mitochondrial targeted fluorescent carbon dot as a mitochondrial fluorescent probe. The mitochondrial targeted fluorescent carbon dot has good mitochondrial targeting ability and can be used for non-damage visualization of mitochondria.

[0006] In order to achieve the above-mentioned purpose, the first aspect of the present invention provides a mitochondrial targeted fluorescent carbon dot, which is prepared from a raw material containing p-aminophenol and an organic solvent; the mitochondrial targeted fluorescent carbon dot is negatively charged; the mitochondrial targeted fluorescent carbon dot is amphiphilic; the emission peak of the mitochondrial targeted fluorescent carbon dot is at 593nm, and its absolute fluorescence quantum yield is 62.06-64.2%.

[0007] Preferably, the particle size of the mitochondria-targeted fluorescent carbon dots is 1.09-5.04 nm, and the average particle size is 2.36-2.93 nm.

[0008] Preferably, the usage ratio of p-aminophenol to the organic solvent is 1 g:720-820 mL.

[0009] Preferably, the organic solvent is selected from one or more of methanol, ethanol, isopropanol and ethylene glycol.

[0010] The second aspect of the present invention provides a method for preparing mitochondria-targeted fluorescent carbon dots, the method comprising the following steps:

[0011] (1) dissolving p-aminophenol in an organic solvent, adjusting the pH value of the obtained mixed solution to 3-5, and then performing a solvothermal reaction to obtain a primary carbon dot product;

[0012] (2) Separating and purifying the initial carbon dot product to obtain a mitochondria-targeted fluorescent carbon dot aqueous solution.

[0013] Preferably, in step (1), the ratio of p-aminophenol to organic solvent is 1 g:720-820 mL.

[0014] Preferably, the organic solvent is selected from one or more of methanol, ethanol, isopropanol and ethylene glycol.

[0015] Preferably, in step (1), the conditions of the solvent thermal reaction include: temperature of 160-200° C. and time of 0.5-5 h.

[0016] Preferably, in step (2), the separation and purification comprises: filtration, extraction and thin layer chromatography separation.

[0017] The third aspect of the present invention provides a mitochondria-targeted fluorescent carbon dot prepared by the method described above.

[0018] A fourth aspect of the present invention provides a use of the mitochondrial-targeted fluorescent carbon dots described above as mitochondrial fluorescent probes.

[0019] Through the above technical solution, the beneficial effects of the present invention are mainly:

[0020] (1) The preparation method of the present invention is simple to operate, the raw materials are cheap and easily available, and the prepared mitochondrial-targeted fluorescent carbon dots are negatively charged.

[0021] (2) The mitochondria-targeted fluorescent carbon dots prepared in the present invention exhibit excellent mitochondrial-targeted imaging and have good mitochondrial-targeted capabilities in different cell lines.

[0022] (3) The mitochondria-targeted fluorescent carbon dots prepared in the present invention have a strong ability to emit fluorescence, and their absolute fluorescence quantum yield reaches 64.2%.

[0023] (4) The mitochondria-targeted fluorescent carbon dots prepared in the present invention exhibit good amphiphilicity and can be dissolved in both lipid media and water.

[0024] (5) The mitochondria-targeted fluorescent carbon dots prepared by the present invention have good biocompatibility and hardly cause any damage to the normal physiological functions of cells after being localized in the mitochondria. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a high performance liquid chromatogram of the mitochondria-targeted fluorescent carbon dots prepared in Example 1;

[0026] Figure 2 is a transmission electron microscope characterization image of the mitochondria-targeted fluorescent carbon dots prepared in Example 1;

[0027] Figure 3 is an atomic force microscope characterization image of the mitochondria-targeted fluorescent carbon dots prepared in Example 1;

[0028] Figure 4 is a Zeta potential characterization diagram of the mitochondria-targeted fluorescent carbon dots prepared in Example 1;

[0029] Figure 5 is a Fourier transform infrared spectrum characterization diagram of the mitochondria-targeted fluorescent carbon dots prepared in Example 1;

[0030] Figure 6 is an X-ray photoelectron spectrum characterization diagram of the mitochondria-targeted fluorescent carbon dots prepared in Example 1;

[0031] Figure 7 are characterization graphs of the absorption spectrum, excitation spectrum and emission spectrum of the mitochondria-targeted fluorescent carbon dots prepared in Example 1;

[0032] Figure 8 These are the fluorescence images of the mitochondria-targeted fluorescent carbon dots prepared in Example 1 co-localized with the mitochondrial probe in HeLa, 4T1 and NIH-3T3 cells;

[0033] Fig. 9This is a detection diagram of the effect of the decrease or disappearance of membrane potential on the mitochondria-targeted fluorescent carbon dots prepared in Example 1 for positioning mitochondria;

[0034] Fig.10 This is a detection diagram of the effects of the mitochondria-targeted fluorescent carbon dots and mitochondrial cationic probes prepared in Example 1 on cell membrane potential and autophagy after imaging. DETAILED DESCRIPTION

[0035] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0036] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0037] The first aspect of the present invention provides a mitochondrial targeted fluorescent carbon dot (C-dots), which is prepared from a raw material containing p-aminophenol and an organic solvent; the mitochondrial targeted fluorescent carbon dot is negatively charged; the mitochondrial targeted fluorescent carbon dot is amphiphilic and can be dissolved in both lipid media and water; the mitochondrial targeted fluorescent carbon dot has a strong ability to emit fluorescence, its emission peak is at 593nm, and its absolute fluorescence quantum yield is 62.06-64.2%.

[0038] In the present invention, in a specific embodiment, the lipid medium can be selected from n-octanol and / or n-butanol.

[0039] In the present invention, in a preferred embodiment, the particle size of the mitochondria-targeted fluorescent carbon dots is 1.09-5.04 nm, and the average particle size is 2.36-2.93 nm.

[0040] In a preferred embodiment, the dosage ratio of p-aminophenol and organic solvent in the mitochondrial targeted fluorescent carbon dots preparation raw material is 1g:720-820mL, for example, 1g:720mL, 1g:730mL, 1g:750mL, 1g:760mL, 1g:770mL, 1g:784mL, 1g:800mL, 1g:810mL, 1g:815mL or 1g:820mL.

[0041] According to the present invention, the organic solvent can be a conventional choice of those skilled in the art. In a specific embodiment, the organic solvent can be selected from one or more of methanol, ethanol, isopropanol or ethylene glycol. In a preferred embodiment, the organic solvent is ethanol.

[0042] The second aspect of the present invention provides a method for preparing mitochondria-targeted fluorescent carbon dots, the method comprising the following steps:

[0043] (1) dissolving p-aminophenol in an organic solvent, adjusting the pH value of the obtained mixed solution to 3-5, and then performing a solvothermal reaction to obtain a primary carbon dot product;

[0044] (2) Separating and purifying the initial carbon dot product to obtain a mitochondria-targeted fluorescent carbon dot aqueous solution.

[0045] In the method described in the present invention, in a specific embodiment, the usage ratio of p-aminophenol and the organic solvent can be 1g:720-820mL, for example, 1g:720mL, 1g:730mL, 1g:750mL, 1g:760mL, 1g:770mL, 1g:784mL, 1g:800mL, 1g:810mL, 1g:815mL or 1g:820mL.

[0046] According to the present invention, the organic solvent can be a conventional choice of those skilled in the art. In a specific embodiment, the organic solvent can be selected from methanol, ethanol, isopropanol or ethylene glycol. In a preferred embodiment, the organic solvent is ethanol.

[0047] In the method described in the present invention, in step (1), before the solvothermal reaction is carried out, the reaction system needs to be in an acidic environment. In a preferred embodiment, the pH value of the mixed solution needs to be adjusted to 3-5. In addition, in order for the solvothermal reaction to proceed effectively and obtain high-performance mitochondrial-targeted fluorescent carbon dots, it needs to be carried out under specific conditions.

[0048] In a specific embodiment, in step (1), a common acidic solution may be used to adjust the pH value of the solution. Preferably, the acidic solution is a sulfuric acid solution and / or a hydrochloric acid solution.

[0049] In a specific embodiment, the temperature of the solvothermal reaction can be 160-200°C, for example 160°C, 170°C, 180°C, 185°C, 190°C, 195°C or 200°C; the time can be 0.5-5h, for example 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h or 5h.

[0050] In the method described in the present invention, the solvent thermal reaction in step (1) specifically includes: after the pH value of the solution is adjusted to 3-5, the solution is evenly transferred to a polytetrafluoroethylene-lined reactor and loaded into a stainless steel reactor jacket; finally, the reactor is placed in a forced air drying oven for heating, and after the reaction is completed, the reactor is cooled to room temperature and the initial carbon dot product is taken out therefrom.

[0051] According to the present invention, in order to obtain high-purity mitochondria-targeted fluorescent carbon dots, the carbon dot primary product needs to be separated and purified.

[0052] In a specific embodiment, in step (2), the separation and purification includes: filtration, extraction and thin layer chromatography separation.

[0053] In a more specific embodiment, the separation and purification of the mitochondrial targeted fluorescent carbon dot primary product comprises:

[0054] S1. Filtration: Filter the carbon dot initial product obtained by the solvent thermal reaction with a 0.22 μm nylon filter to remove large particle impurities;

[0055] S2, extraction: first, the solvent in the filtered product is completely evaporated by rotary evaporation, water is added to dissolve, and then filtered, and then butyl acetate is added thereto and continuously shaken and extracted until the reddish brown extract turns colorless, and finally, all the extracts are concentrated by a rotary evaporator;

[0056] S3. Thin layer chromatography separation: The concentrated extract is separated by preparative thin layer chromatography (PTLC), and then eluted by column chromatography to obtain a mitochondria-targeted fluorescent carbon dot aqueous solution.

[0057] In a specific embodiment, the conditions for rotary evaporation concentration during the extraction process can be selected within a wide range. In order to further improve the purity of mitochondrial targeted fluorescent carbon dots, in a preferred embodiment, the rotary evaporation concentration temperature is 55-70°C, for example, 55°C, 58°C, 60°C, 62°C, 65°C, 68°C or 70°C.

[0058] In the present invention, the conditions of thin layer chromatography can be selected in a wide range. In order to further improve the purity of mitochondrial targeted fluorescent carbon dots, under the preferred conditions, the thin layer chromatography developing agent is a mixed solution of n-hexane and ethyl acetate in a volume ratio of 1:2-4.

[0059] In the method of the present invention, under preferred conditions, the column chromatography eluent is a mixed solution of n-hexane and ethyl acetate in a volume ratio of 1:2-8.

[0060] In a specific embodiment, the rotary evaporation drying conditions in the thin layer chromatography can be selected within a wide range. In order to improve the drying effect and effectively remove the organic solvent, in a preferred embodiment, the rotary evaporation drying temperature is 35-50°C, for example 35°C, 38°C, 40°C, 42°C, 45°C, 48°C or 50°C.

[0061] In the method of the present invention, in order to facilitate the preservation of the prepared mitochondria-targeted fluorescent carbon dots and reduce losses, under preferred conditions, after column chromatography treatment, the product needs to be redispersed in water and filtered to obtain a pure mitochondria-targeted fluorescent carbon dot aqueous solution.

[0062] The third aspect of the present invention provides a mitochondrial targeted fluorescent carbon dot prepared by the method described above. The mitochondrial targeted fluorescent carbon dot is prepared from a raw material containing p-aminophenol and an organic solvent; the mitochondrial targeted fluorescent carbon dot is negatively charged; the mitochondrial targeted fluorescent carbon dot is amphiphilic and can be dissolved in both lipid media and water; the mitochondrial targeted fluorescent carbon dot has a strong ability to emit fluorescence, its emission peak is at 593nm, and its absolute fluorescence quantum yield is 62.06-64.2%.

[0063] A fourth aspect of the present invention provides a use of the mitochondrial-targeted fluorescent carbon dots described above as mitochondrial fluorescent probes.

[0064] The experiments of co-localization of mitochondria-targeted fluorescent carbon dots and mitochondrial blue probes in the examples show that the mitochondria-targeted fluorescent carbon dots provided by the present invention have good mitochondrial targeting capabilities in different cell lines (including cancer cell lines and normal cell lines).

[0065] The present invention will be described in detail below by way of examples. Unless otherwise specified, all methods are conventional in the art. The experimental materials used in the following examples, unless otherwise specified, can be purchased from reagent suppliers.

[0066] Example 1

[0067] (1) 0.0982 g of p-aminophenol was dissolved in 77 mL of ethanol (the ratio of p-aminophenol to ethanol was 1 g:784 mL), and then the pH value of the obtained mixed solution was adjusted to 4 with sulfuric acid solution. The solution was then transferred to a 25 mL reactor and loaded into a stainless steel reactor jacket. Finally, the reactor was placed in a forced air drying oven and heated at 180°C for 1 h. After the reaction was completed, the reactor was naturally cooled to room temperature, and the carbon dot primary product was taken out from the reactor.

[0068] (2) filtering the initial carbon dot product using a 0.22 μm nylon filter;

[0069] (3) The filtered solvent was completely evaporated by rotary evaporation (concentration temperature was 65°C), about 15 mL of water was added for dissolution, and then filtered, and then 20 mL of butyl acetate was added thereto and continuously shaken for extraction until the reddish brown extract turned into colorless, and finally, all the extracts were concentrated to about 5 mL by rotary evaporator (concentration temperature was 65°C) for subsequent separation;

[0070] (4) The concentrated extract was separated by preparative thin layer chromatography (PTLC). Specifically, the concentrated extract was dropped onto a silica gel plate. After the solvent on the silica gel plate was completely evaporated, the silica gel plate was placed in a developing tank pre-added with a developing agent (a mixed solution of n-hexane and ethyl acetate in a volume ratio of 3:8) for thin layer chromatography separation. After 50 minutes, the silica gel plate was taken out and dried. Then, the silica gel powder that produced orange fluorescent bands under the 365nm ultraviolet band was collected, and then the components in the silica gel powder were eluted with a silica gel column (the eluent was a mixed solution of n-hexane and ethyl acetate in a volume ratio of 1:4). Finally, the solution eluted from the silica gel column was dried by rotary evaporation (40°C) to remove the organic solvent, and then the product was redispersed in water and filtered with a 0.22μm mixed cellulose ester filter to obtain a pure mitochondrial targeted fluorescent carbon dot aqueous solution.

[0071] Example 2

[0072] The method of Example 1 was followed, except that the ratio of p-aminophenol to ethanol was 1 g:720 mL.

[0073] Example 3

[0074] The method of Example 1 was followed, except that the ratio of p-aminophenol to ethanol was 1 g:820 mL.

[0075] Example 4

[0076] The method of Example 1 was followed, except that the solvent thermal reaction conditions included: temperature of 160° C. and time of 5 h.

[0077] Example 5

[0078] The method of Example 1 was followed, except that the solvent thermal reaction conditions included: temperature of 200° C. and time of 0.5 h.

[0079] Comparative Example 1

[0080] According to the method of patent application CN108504349A, the specific preparation method is as follows:

[0081] (1) Dissolve 30 mg of citric acid and 14.8 mg of 8-hydroxyjulolidine in 2 mL of distilled water and place in a microwave reaction tube;

[0082] (2) The mitochondria-targeted fluorescent carbon dots were obtained by microwave-assisted hydrothermal reaction at 160 °C for 2 h, then cooled to room temperature (25 °C), filtered and vacuum freeze-dried.

[0083] The mitochondria-targeted fluorescent carbon dots prepared in Comparative Example 1 have positive charges.

[0084] Comparative Example 2

[0085] According to the method of patent application CN106833629A, the specific preparation method is as follows:

[0086] (1) dissolving 1 g of chitosan, 200 μL of ethylenediamine and 500 mg of mercaptosuccinic acid in 0.4 mol / L dilute hydrochloric acid to obtain a mixed solution;

[0087] (2) reacting the mixed solution obtained in step (1) in a hydrothermal reactor at 180° C. for 24 h to obtain a product;

[0088] (3) Centrifuge or filter the product obtained in step (2) to obtain a mitochondrial targeted fluorescent carbon dot solution.

[0089] The mitochondria-targeted fluorescent carbon dots prepared in Comparative Example 1 have significant positive charges and are suitable for mitochondrial imaging in aqueous biological systems.

[0090] Test Example 1

[0091] (1) The mitochondria-targeted fluorescent carbon dots prepared in Example 1 were detected by high performance liquid chromatography (HPLC, Thermo Scientific UltiMate 3000). The results are as follows: Figure 1 As shown, the component contains only a single chromatographic peak (8.446min). The results show that the separation effect is good and a highly purified component is obtained by the combined separation system.

[0092] (2) The particle sizes of the mitochondria-targeted fluorescent carbon dots prepared in Examples 1-5 and Comparative Examples 1-2 were tested using a transmission electron microscope (TEM, Hitachi HT-7800). The results are shown in Table 1. Figure 2 shown.

[0093] Table 1

[0094] serial number Particle size range (nm) Average particle size (nm) Example 1 1.09-4.36 2.36 Example 2 1.79-4.28 2.88 Example 3 1.52-4.81 2.92 Example 4 1.83-4.35 2.57 Example 5 1.68-5.04 2.93 Comparative Example 1 1.1-3.4 2.03 Comparative Example 2 0.8-3.9 2.10

[0095] (3) Atomic force microscopy (AFM, Bruker Dimension Icon) was used to characterize the thickness of the mitochondria-targeted fluorescent carbon dots prepared in Example 1. The results are as follows: Figure 3As shown, the thickness of the carbon dots is distributed in the range of 1.4-3.1 nm, indicating that the morphology of the carbon dots is spherical / quasi-spherical.

[0096] (4) The Zeta potential of the mitochondria-targeted fluorescent carbon dots prepared in Examples 1-5 and Comparative Example 2 in an aqueous phase (pH 6.6-7.4) was tested using a dynamic light scattering instrument (Malvern ZS90 Zeta sizer). The results are shown in Table 2. Figure 4 shown.

[0097] (5) The absorption spectrum, excitation spectrum and emission spectrum of the mitochondria-targeted fluorescent carbon dots prepared in Example 1 were characterized using an ultraviolet-visible spectrophotometer (Hitachi U-2910) and a fluorescence spectrometer (Hitachi F-4600). Figure 7 As shown, from Figure 7 It can be seen that the mitochondrial targeted fluorescent carbon dots have a characteristic absorption peak at 566nm in addition to the typical C=C π-π* and CN / CO / C=O n-π* transition absorption at 238nm and 286nm; under different wavelengths of excitation light, its emission peak is around 593nm, showing an emission that is not dependent on excitation. In addition, the absolute fluorescence quantum yield of the mitochondrial targeted fluorescent carbon dots prepared in Examples 1-5 and Comparative Example 2 was detected using a steady-state fluorescence spectrometer (Edinburgh FLS 1000), and the results are shown in Table 2.

[0098] Table 2

[0099] serial number Zeta potential (mV) Absolute fluorescence quantum yield / % Example 1 -2～-6 64.2 Example 2 -3～-6 62.06 Example 3 -3～-5 63.24 Example 4 -2～-5 63.09 Example 5 -2～-4 63.38 Comparative Example 2 28.1 11.8

[0100] It can be seen from the results in Table 2 that the mitochondria-targeted fluorescent carbon dots prepared in Examples 1-5 are negatively charged, and their absolute fluorescence quantum yields are 62.06-64.2%.

[0101] Test Example 2

[0102] The n-octanol / water partition coefficient (lgP) of the mitochondrial targeted fluorescent carbon dots obtained in Examples 1-5 was determined by the shaking bottle method. The method is as follows: the ratio of n-octanol to water is controlled to 1:4, 4 mL of mitochondrial targeted fluorescent carbon dot aqueous solution (102.67 μg / mL, pre-saturated with n-octanol) is taken in a 10 mL centrifuge tube, 1 mL of n-octanol (pre-saturated with water) is added, and the centrifuge tube is placed in a vortex mixer for 0.5 h, and then allowed to stand for 24 h to allow the two phases to separate; the aqueous phase is drawn with a needle syringe to avoid contamination of the n-octanol phase, and the absorbance of the aqueous phase is measured by UV-Vis, and the results are shown in Table 3.

[0103] Table 3

[0104] serial number Equilibrium concentration in n-octanol (μg / mL) n-octanol / water partition coefficient (lgP) Example 1 368.36 1.54 Example 2 363.38 1.49 Example 3 373.33 1.60 Example 4 365.87 1.51 Example 5 370.84 1.57

[0105] From the results in Table 3, it can be seen that the 1gP of the mitochondria-targeted fluorescent carbon dots prepared in Examples 1-5 is 1.49-1.60, showing good amphiphilicity (0<1gP<4), and can be dissolved in both lipid media and aqueous phases.

[0106] Test Example 3

[0107] (1) Fourier transform infrared spectrometer (FTIR, Buker INVENIO S-type) was used to characterize the surface functional groups of the mitochondrial targeted fluorescent carbon dots prepared in Example 1. Figure 5 It can be seen that in the infrared spectrum, 3150-3685cm -1 The broad absorption bands are due to the stretching vibrations of OH and NH. In addition, there are also broad absorption bands at 2798-2987, 1520-1803, 1288-1520 and 970-1220 cm -1 The four absorption bands were respectively attributed to the stretching vibration of CH, C=C, CN and the in-plane bending vibration of CO stretching vibration / OH. Infrared spectra showed that the surface of mitochondria-targeted fluorescent carbon dots may retain the hydroxyl and amino groups in p-aminophenol.

[0108] (2) X-ray photoelectron spectroscopy (XPS, Thermo Scientific K-Alpha) was used to characterize the chemical composition of the mitochondria-targeted fluorescent carbon dots prepared in Example 1. Figure 6 It can be seen that the spectrum shows three typical peaks at ~285eV, ~399eV, and ~533eV, corresponding to C, N, and O, respectively, and the percentages of the three atoms are 68.58%, 5.43%, and 25.99%, respectively, indicating that the prepared mitochondria-targeted fluorescent carbon dots are composed of C, N, and O.

[0109] Test Example 4

[0110] A laser confocal microscope (CLSM, Leica TCS SP8) was used to capture the fluorescence image of the co-localization of the mitochondria-targeted fluorescent carbon dots and the organelle probe obtained in Example 1. The method is as follows:

[0111] (1) HeLa cells (5×10 4 / mL) were inoculated into confocal microplates and cultured for 24h;

[0112] (2) Wash the cells three times with phosphate-buffered saline (PBS), add 1 mL of 1640 medium containing 20 μg / mL mitochondria-targeted fluorescent carbon dots, and incubate for 4 h;

[0113] (3) After washing the cells three times with PBS, add 1 mL of mitochondrial blue probe (MitoLite TM Blue FX490, MLB, take 20 μL of 500×MLB and dilute to 10 mL with 1640 medium) and incubate for another 90 min in 1640 medium;

[0114] (4) Wash the cells three times with PBS and add 1 mL of PBS for subsequent imaging.

[0115] In the lysosome co-localization experiment, after washing away the mitochondria-targeted fluorescent carbon dots, 1 mL of lysosome green probe (LysoTracker Green DND-26, LTG, 100 nM) was added and incubated for 20 min, and the rest of the steps were the same as above.

[0116] In the nuclear co-localization experiment, after washing away the mitochondria-targeted fluorescent carbon dots, the cells were treated with 4% paraformaldehyde at room temperature for 20 min, and after washing with PBS, 1 mL of nuclear dye DAPI reagent (1 μg / mL) was added and incubated in the dark for 5 min. The rest of the steps were the same as above. Fluorescence imaging of the cells was performed using a 63× oil lens of CLSM.

[0117] Depend on Figure 8 It can be seen that the fluorescence of mitochondrial targeted fluorescent carbon dots and mitochondrial probes in HeLa cells almost completely overlapped. The Pearson correlation coefficient (PCC) and overlap coefficient (OLC) were 0.91 and 0.93 respectively; in addition, the possibility of mitochondrial targeted fluorescent carbon dots for imaging mitochondria in different cells was also tested on other cancer cell lines (4T1 cells) and normal cell lines (NIH-3T3 cells). The results showed that the PCC and OLC of mitochondrial targeted fluorescent carbon dots and mitochondrial probes were both higher than 0.8. Combined with the Plotprofile graph and the Scatter plot graph, it was shown that mitochondrial targeted fluorescent carbon dots had good mitochondrial targeting ability in different cell lines (including cancer cell lines and normal cell lines).

[0118] Test Example 5

[0119] The effect of the decrease or disappearance of membrane potential on the mitochondrial targeting fluorescent carbon dots obtained in Example 1 was tested. The method is as follows: HeLa cells were treated with 10 μM CCCP (carbonyl cyanide-3-chlorophenylhydrazone, an oxidative phosphorylation uncoupler) for 30 minutes in advance, and then incubated with mitochondrial targeting fluorescent carbon dots and Rho123 (a probe for detecting MMP) for a period of time, and the changes in fluorescence intensity of cells treated with and without CCCP were compared. The results are shown in Figure 2. Fig. 9As shown in the figure, compared with the control group, the fluorescence of Rho123 was greatly weakened after CCCP treatment, while the fluorescence intensity of mitochondria-targeted fluorescent carbon dots remained almost unchanged, showing excellent mitochondrial targeting ability independent of MMP. In addition, after treatment with 4% paraformaldehyde for 20 minutes, the MMP of fixed cells completely disappeared, which can be reflected by the almost complete disappearance of the fluorescence intensity of Rho123. However, the fluorescence of mitochondria-targeted fluorescent carbon dots can still be observed after the disappearance of MMP. This shows that the targeting of mitochondria-targeted fluorescent carbon dots to mitochondria does not rely on electrostatic adsorption. Since mitochondria-targeted fluorescent carbon dots are amphiphilic, the hydrophobic structure and hydrophilic groups on their surface may play a certain role, such as the hydrophobic interaction between the hydrophobic structure on the mitochondria-targeted fluorescent carbon dots and hydrophobic molecules such as phospholipids on the mitochondrial membrane, and the hydrogen bonding between surface hydrophilic groups such as amino and hydroxyl groups and mitochondrial proteins.

[0120] Test Example 6

[0121] The effects of the mitochondria-targeted fluorescent carbon dots and mitochondrial cationic probes obtained in Example 1 on cell membrane potential and autophagy were tested. The method is as follows: 4T1 cells were treated with mitochondria-targeted fluorescent carbon dots (20 μg / mL) and mitochondrial cationic probes (MLB) respectively, and MMP was detected with Rho123 after incubation for 6 hours. Fig.10 It can be seen that the fluorescence intensity of Rho123 did not decrease significantly after treatment with mitochondria-targeted fluorescent carbon dots, which means that MMP remained almost unchanged. However, the fluorescence intensity decreased significantly after treatment with cationic probes, indicating that MMP was significantly reduced. Moreover, after treatment with cationic probes, many mitochondria changed from healthy filaments to abnormal dot-like / fragmented shapes, while no obvious changes were observed in cells treated with mitochondria-targeted fluorescent carbon dots. This proves that mitochondria-targeted fluorescent carbon dots have little effect on MMP and mitochondrial function. In addition, the MDC (autophagy detection reagent) method was used to detect whether 4T1 cells would produce autophagy after long-term treatment with mitochondria-targeted fluorescent carbon dots (1 μg / mL) and mitochondrial cationic dyes (Mito Tracker Red, MTR, 1 μg / mL). The results showed that the incubation time of mitochondria-targeted fluorescent carbon dots was extended to 36 hours without producing autophagic vesicles, while the cells underwent obvious autophagy after 24 hours of MTR treatment, further indicating that mitochondria-targeted fluorescent carbon dots have little damage to their normal physiological functions after positioning mitochondria.

[0122] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A mitochondrial-targeted fluorescent carbon dot, characterized in that: The mitochondrial targeted fluorescent carbon dots are prepared from raw materials containing p-aminophenol and an organic solvent; the mitochondrial targeted fluorescent carbon dots are negatively charged; the mitochondrial targeted fluorescent carbon dots are amphiphilic; the emission peak of the mitochondrial targeted fluorescent carbon dots is at 593 nm, and the absolute fluorescence quantum yield thereof is 62.06-64.2%; Wherein, the particle size of the mitochondria-targeted fluorescent carbon dots is 1.09-5.04 nm, and the average particle size is 2.36-2.93 nm; The dosage ratio of p-aminophenol to organic solvent is 1g:720-820mL; The organic solvent is ethanol; The preparation of the mitochondria-targeted fluorescent carbon dots comprises the following steps: (1) dissolving p-aminophenol in ethanol, adjusting the pH value of the resulting mixed solution to 3-5, and then performing a solvothermal reaction to obtain a primary carbon dot product; (2) separating and purifying the initial carbon dot product to obtain a mitochondria-targeted fluorescent carbon dot aqueous solution; Wherein, in step (1), the conditions of the solvent thermal reaction include: temperature of 160-200°C and time of 0.5-5h.

2. A method for preparing mitochondria-targeted fluorescent carbon dots, characterized in that: The method comprises the following steps: (1) dissolving p-aminophenol in ethanol, adjusting the pH value of the resulting mixed solution to 3-5, and then performing a solvothermal reaction to obtain a primary carbon dot product; (2) separating and purifying the initial carbon dot product to obtain a mitochondria-targeted fluorescent carbon dot aqueous solution; Wherein, in step (1), the conditions of the solvent thermal reaction include: temperature of 160-200°C and time of 0.5-5h.

3. The method according to claim 2, characterized in that In step (1), the dosage ratio of p-aminophenol to ethanol is 1 g:720-820 mL.

4. The method according to claim 2 or 3, characterized in that: In step (2), the separation and purification includes: filtration, extraction and thin layer chromatography separation.

5. Mitochondria-targeted fluorescent carbon dots prepared by the method according to any one of claims 2 to 4.

6. Use of the mitochondrial-targeted fluorescent carbon dots described in claim 1 or 5 as mitochondrial fluorescent probes.

Images