Copper-doped mitochondrial-targeted carbon dots, preparation method and application thereof

Abstract

The present invention belongs to the technical field of nano fluorescent materials, and specifically relates to a copper-doped mitochondrial targeted carbon dot, a preparation method and its application. The preparation method of the fluorescent carbon dot comprises the following steps: step 1): adding a carbon source, a copper source and a nitrogen source to a sulfuric acid solution in sequence, and mixing to obtain a mixed solution; the mass ratio of the carbon source, the copper source and the nitrogen source is 1-15:1-23:1-20; step 2): placing the mixed solution under high temperature reaction; step 3): centrifuging the solution obtained in step 2), collecting the upper solution for filtration, dialysis, collecting a solution with a molecular weight of more than 800Da, and freeze-drying to obtain a copper-doped mitochondrial targeted carbon dot. The synthesis process of the fluorescent carbon dot provided by the present invention is simple and convenient, with high yield, and the prepared fluorescent carbon dot can target mitochondria, and can spontaneously emit red fluorescence, which is helpful for conducting small animal in vivo experiments, and has a certain killing effect on brain glioma cells.

Description

Technical Field

[0001] The invention belongs to the technical field of nano fluorescent materials, and specifically relates to a preparation method and application of copper-doped mitochondrial targeted carbon dots. Background Art

[0002] Carbon dots (or carbon quantum dots, carbon nanodots) are a type of sp 2 Quasi-spherical nanoparticles formed by the polymerization of graphite core or amorphous carbon. Carbon dots have excellent optical / electrical properties and rich surface functional groups. They are widely used in biochemical sensing, display and photoelectrocatalysis. In addition, excellent biocompatibility also strongly promotes the application of carbon dots in the field of nanobiomedicine. The preparation method of carbon dots is simple, the source of raw materials is abundant and the cost is low. At present, the preparation of carbon dots is mainly top-down and bottom-up. The top-down method mainly uses arc discharge, laser ablation and electrochemical oxidation to reduce the size of carbon precursors such as graphite, graphene or carbon nanotubes that have perfect sp2 structure but lack effective band gap to emit fluorescence to obtain nano-scale particles. The bottom-up method is to prepare carbon dots through a series of reactions such as high temperature dehydration and carbonization by using solvothermal and microwave synthesis techniques, with small molecules as precursors. Because of its simple preparation process, it has developed into the mainstream preparation method of carbon dots.

[0003] Copper ions also have certain antibacterial properties, but they need to work at higher concentrations. Studies have shown that divalent copper ions will react with hydrogen peroxide under weakly acidic conditions to produce hydroxyl radicals. Copper-doped carbon dots are an effective photocatalyst that produces reactive oxygen radicals under visible light irradiation. Studies have shown that copper-doped carbon dots have a photocatalytic bactericidal effect on Escherichia coli. However, the current preparation of copper-doped carbon dots has the following problems: the existing copper-doped carbon dots mostly use cysteine ​​as a raw material to synthesize carbon dots during preparation. In order to make the synthesized copper-doped carbon dot nanomaterials have red light, a specific fluorescent dye needs to be added. This type of synthesis method is relatively complex and cumbersome, and it cannot spontaneously emit red fluorescence. Therefore, it is urgent to develop a preparation method for copper-doped carbon dots that has a simple synthesis method and can spontaneously emit red fluorescence without the use of red fluorescent reagents. Summary of the invention

[0004] In order to solve the problem that the synthesis method in the prior art is relatively complicated and cumbersome and cannot spontaneously emit red fluorescence, the present invention provides a copper-doped mitochondrial-targeted carbon dot, a preparation method and an application thereof.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The present invention provides a method for preparing copper-doped mitochondria-targeted carbon dots, comprising the following steps:

[0007] Step 1): adding a carbon source, a copper source, and a nitrogen source to a sulfuric acid solution in sequence, and mixing them to obtain a mixed solution;

[0008] The mass ratio of the carbon source, the copper source and the nitrogen source is 1-15:1-23:1-20;

[0009] Step 2): placing the mixed solution under high temperature for reaction;

[0010] Step 3): centrifuge the solution obtained in step 2), collect the upper solution, filter and dialyze, collect the solution with a molecular weight of more than 800 Da, and freeze-dry to obtain copper-doped mitochondrial targeted carbon dots.

[0011] Preferably, the nitrogen source is arginine, the carbon source is o-phenylenediamine, and the copper source is copper nitrate; and the concentration of the sulfuric acid solution is 3.4-3.6 mol / L.

[0012] Preferably, the mass ratio of o-phenylenediamine, copper nitrate and arginine is 1-9:3-17:2-15.

[0013] Preferably, in step 2), the mixed solution is reacted at a high temperature of 160° C.-220° C. for 6 to 24 hours.

[0014] Preferably, in step 3), the solution obtained in step 2) is centrifuged at 3000 rpm for 3 to 5 min.

[0015] Preferably, in step 3), the upper layer solution is filtered and then placed in a 800Da dialysis bag for 45 to 50 hours in the dark.

[0016] Preferably, in step 3), the solution in the dialysis bag after dialysis is freeze-dried at -60°C for more than 72 hours and stored away from light.

[0017] The present invention also provides copper-doped mitochondrial targeted carbon dots prepared by a method for preparing copper-doped mitochondrial targeted carbon dots.

[0018] The present invention also provides the use of copper-doped mitochondria-targeted carbon dots in the preparation of drugs for treating brain gliomas.

[0019] The present invention also provides the use of copper-doped mitochondria-targeted carbon dots in the preparation of living imaging agents.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The synthesis process of the copper-doped carbon dots provided by the present invention is simple and convenient, with high yield, and the prepared copper-doped carbon dots have the advantages of good stability and good biocompatibility; at the same time, the prepared copper-doped carbon dots can spontaneously emit red fluorescence, can target mitochondria, are helpful for conducting small animal in vivo experiments, and have a certain killing effect on brain glioma cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the ultraviolet absorption peak of the copper-doped mitochondria-targeted carbon dots prepared in Example 1 of the present invention;

[0023] Figure 2 is the ultraviolet absorption peak of the copper-doped mitochondria-targeted carbon dots prepared in Example 2 of the present invention;

[0024] Figure 3 is the ultraviolet absorption peak of the copper-doped mitochondria-targeted carbon dots prepared in Example 3 of the present invention;

[0025] Figure 4 This is a fluorescence spectrum of the copper-doped mitochondria-targeted carbon dots prepared in Example 1 of the present invention;

[0026] Figure 5 is a transmission electron microscopy image of the copper-doped mitochondria-targeted carbon dots prepared in Example 1 of the present invention, wherein A is a transmission electron microscopy particle size image, and B is a crystal lattice;

[0027] Figure 6 is a lattice diagram of the copper-doped mitochondria-targeted carbon dots prepared in Example 1 of the present invention;

[0028] Figure 7 The potential distribution diagram of the copper-doped mitochondria-targeted carbon dots prepared in Example 1 of the present invention, wherein A is the cell phagocytosis diagram, B is Mito Tracker Green, C is Cu-CD-NSs, and D is the Merge diagram;

[0029] Figure 8 The experimental results of cellular uptake and mitochondrial targeting of the copper-doped mitochondrial targeted carbon dots prepared in Example 1 of the present invention, wherein A is a cell phagocytosis image, B is a mitochondrial fluorescence staining image, C is a material-targeted mitochondrial fluorescence image, and D is a co-localization double staining image;

[0030] Fig. 9 This is the in vivo imaging of small animals using the copper-doped mitochondria-targeted carbon dots prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0031] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0032] Example 1

[0033] The preparation of copper-doped mitochondrial targeted carbon dots includes the following raw materials: o-phenylenediamine, copper nitrate, arginine; and sulfuric acid solution is used as a solvent.

[0034] The preparation method of the copper-doped mitochondria-targeted carbon dots is as follows:

[0035] Step 1: Add 0.1 g of o-phenylenediamine, 0.3 g of copper nitrate, and 0.2 g of arginine to a 3.5 mol / L sulfuric acid solution in sequence, and completely dissolve and mix them by ultrasonication to obtain a mixed solution.

[0036] Step 2: Pour the mixed solution into a polytetrafluoroethylene high temperature reactor and react at 190°C for 15 hours.

[0037] Step 3: Centrifuge the solution obtained in step 2 at 3000 rpm for 3 min, collect the upper solution, filter it through a filter membrane with a pore diameter of 0.22 μM, place the filtrate in an 800 Da dialysis bag and dialyze it in deionized water in the dark for 48 hours, collect the solution in the dialysis bag and freeze-dry it at -60°C to obtain a copper-doped mitochondrial-targeted carbon dot solid.

[0038] Step 4: Dissolve the copper-doped mitochondria-targeted carbon dots solid in water and measure the absorption peak of the carbon dots using a UV-visible spectrophotometer (see Figure 1 ).

[0039] Example 2

[0040] The preparation of copper-doped mitochondrial targeted carbon dots includes the following raw materials: o-phenylenediamine, copper nitrate, arginine; and sulfuric acid solution is used as a solvent.

[0041] The preparation method of the copper-doped mitochondria-targeted carbon dots is as follows:

[0042] Step 1: Add 0.5 g of o-phenylenediamine, 1.0 g of copper nitrate, and 0.85 g of arginine to a 3.5 mol / L sulfuric acid solution in sequence, and completely dissolve and mix them by ultrasonication to obtain a mixed solution.

[0043] Step 2: Pour the mixed solution into a polytetrafluoroethylene high temperature reactor and react at 160°C for 6 hours.

[0044] Step 3: Centrifuge the solution obtained in step 2 at 3000 rpm for 5 min, collect the upper solution, filter it through a filter membrane with a pore diameter of 0.22 μM, place the filtrate in an 800 Da dialysis bag and dialyze it in deionized water in the dark for 45 hours, collect the solution in the dialysis bag and freeze-dry it at -60°C to obtain a copper-doped mitochondrial-targeted carbon dot solid.

[0045] Step 4: Dissolve the copper-doped mitochondria-targeted carbon dots solid in water and measure the absorption peak of the carbon dots using a UV-visible spectrophotometer (see Figure 2 ).

[0046] Example 3

[0047] The preparation of copper-doped mitochondrial targeted carbon dots includes the following raw materials: o-phenylenediamine, copper nitrate, arginine; and sulfuric acid solution is used as a solvent.

[0048] The preparation method of the copper-doped mitochondria-targeted carbon dots is as follows:

[0049] Step 1: Add 0.9 g of o-phenylenediamine, 1.7 g of copper nitrate, and 1.5 g of arginine to a 3.5 mol / L sulfuric acid solution in sequence, and completely dissolve and mix them by ultrasonication to obtain a mixed solution.

[0050] Step 2: Pour the mixed solution into a polytetrafluoroethylene high temperature reactor and react at 220°C for 24 hours.

[0051] Step 3: Centrifuge the solution obtained in step 2 at 3000 rpm for 5 min, collect the upper solution, filter it through a filter membrane with a pore diameter of 0.22 μM, place the filtrate in an 800 Da dialysis bag and dialyze it in deionized water in the dark for 50 hours, collect the solution in the dialysis bag and freeze-dry it at -60°C to obtain a copper-doped mitochondrial targeted carbon dot solid.

[0052] Step 4: Dissolve the copper-doped mitochondria-targeted carbon dots solid in water and measure the absorption peak of the carbon dots using a UV-visible spectrophotometer (see Figure 3 ).

[0053] Comparative Example 1

[0054] A method for preparing copper-doped mitochondria-targeted carbon dots is as follows:

[0055] Step 1: o-phenylenediamine (300 mg) and L-cysteine ​​(675 mg) were dissolved in 30 mL of sodium hydroxide (NaOH) solution (0.2 M, to increase the solubility of L-cysteine).

[0056] Step 2: The solution obtained in step 1 was transferred to a polytetrafluoroethylene reactor and subjected to a hydrothermal process at 160° C. for 10 hours to obtain an orange-yellow CDs solution.

[0057] Step 3: CuNO3 solution (60 mmol) was mixed with the synthesized CDs solution (containing 50 mg CDs), and the resulting mixture was kept at 25°C for 2 hours without stirring to obtain Cu-CD-NSs.

[0058] Step 4: In order to improve the stability of Cu-CD-NSs, 3 mL of Cu-CD-NSs solution and 9 mg of thiol-PEG2k were mixed, and the reaction was stirred at 25°C overnight (overnight time > 12 h) to obtain a PEG-modified Cu-CD-NSs solution; the PEG-modified Cu-CD-NSs solution was then dialyzed with deionized water for 48 hours.

[0059] Step 5: A mixture of 1.0 mg HS-PEG2k-NH2 and 0.125 mg / 0.268 mg FITC / RITC was reacted in sodium carbonate buffer (100 mM, pH 9.5) at 25°C overnight, and then dialyzed for next use; for 3 mL Cu-CD-NSs solution, 8.55 mg HS-PEG2k and 0.51 mg HS-PEG2k-FITC / 0.57 mg HS-PEG2k-RITC were added, and the obtained mixture was stirred at 25°C for 24 hours and dialyzed against dimethyl sulfoxide and deionized water to finally obtain copper-doped mitochondrial targeted carbon dots.

[0060] The absorption peak of the copper-doped mitochondrial targeted carbon dots prepared in Example 1-3 was measured by UV-visible spectrophotometer. Figure 1-3 The measured absorption peaks are around 615nm, 614nm, and 617nm, and the maximum emission peak is 635nm. The fluorescence spectrum is shown in Figure 4 .

[0061] Depend on Figure 4 It can be seen that the fluorescence of the copper-doped mitochondrial-targeted carbon dots prepared in the present invention is red fluorescence (the wavelength range of the red fluorescence is: 780-620 nm).

[0062] Compared with Comparative Example 1, the copper-doped mitochondrial-targeted carbon dots prepared by the present invention have a simple and convenient synthesis process, are highly soluble in water, have good stability, and can spontaneously emit red fluorescence for later use in in vivo imaging.

[0063] Taking Example 1 of the present invention as an example, the performance and application of the copper-doped mitochondrial targeted carbon dots provided by the present invention are further described below.

[0064] 1. Transmission electron microscopy and potential measurement of the copper-doped mitochondrial targeted carbon dots prepared in Example 1

[0065] The copper-doped mitochondrial-targeted carbon dots prepared in Example 1 were weighed, deionized water was added to make a concentration of 1 mg / ml, and the size and lattice of the particles were photographed using a high-resolution electron microscope; the 1 mg / ml carbon dot solution was added to a Malvern potential sample cell, and the particles were measured using a Malvern laser particle size analyzer.

[0066] Transmission electron microscopy and potential results are shown in Figure 1-3 , transmission electron microscopy measured that the particle size of copper-doped mitochondrial-targeted carbon dots was about 3 nm; the ζ potential was around +16 mv, proving that positive potential can better target mitochondria.

[0067] 2. Cellular uptake and mitochondrial targeting

[0068] 5×10 LN229 glioma cells were plated in six-well plates. 5 After incubation for 24 hours, the aqueous solution of copper-doped mitochondria-targeted carbon dots obtained in step 4 of Example 1 was added, incubated for 4 hours, washed three times with 0.01M PBS solution, and then the mitochondrial green fluorescent dye Mito Tracker Green was added and incubated for 30 minutes. After washing three times with PBS solution, the cells were observed under a fluorescence microscope. The experimental results are shown in FIG. Figure 7 .

[0069] 3. Cell Viability Assay

[0070] Glioma cells LN229 were plated in 96-well plates, 7000 cells / well, and incubated for 24 hours. Then, the concentration gradient of 0, 1, 2, 4, 8, 16, 32, 64, and 128 (μg / ml) was added to each well and incubated for 24 hours. CCK-8 reagent was then added and incubated for 1 hour before measuring cell viability. The experimental results are shown in Figure 8 .

[0071] Depend on Figure 8 It can be seen that the cell viability of glioma LN229 cells is less than 10% at 128 μg / ml, indicating that the copper-doped mitochondria-targeted carbon dots prepared by the present invention can successfully inhibit the viability of glioma cells. In order to further illustrate the application of the copper-doped mitochondria-targeted carbon dots provided by the present invention, the copper-doped mitochondria-targeted carbon dots prepared in Example 1 are taken as an example and applied to small animal in vivo experiments. The specific operation is as follows:

[0072] The copper-doped mitochondrial-targeted carbon dots prepared in Example 1 were diluted with physiological saline to a 1 mg / ml solution, and then the solution was injected into nude mice through the tail vein. After 8 hours, a small animal imaging device was used to capture fluorescent images. The in vivo imaging of the small animals was as follows: Fig. 9 shown.

[0073] Depend on Fig. 9 It can be seen that the copper-doped mitochondrial-targeted carbon dots prepared in the present application have a red fluorescence emission wavelength of about 635 nm. The red fluorescence has good penetration in the body and can be used for in vivo experiments on small animals and later in diagnostic technology.

[0074] The present invention provides a method for preparing copper-doped mitochondrial targeted carbon dots, wherein the synthesis process is simple and convenient. The prepared copper-doped mitochondrial targeted carbon dots can spontaneously emit red light and the fluorescence is not easily quenched, can successfully target mitochondria, and can induce brain glioma cell death.

[0075] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0076] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0077] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing copper-doped mitochondrial-targeted carbon dots, characterized in that: The steps include: Step 1): Add a carbon source, a copper source, and a nitrogen source to a sulfuric acid solution in sequence, and mix well to obtain a mixed solution; The nitrogen source is arginine, the carbon source is o-phenylenediamine, and the copper source is copper nitrate; the concentration of the sulfuric acid solution is 3.4-3.6 mol / L; The mass ratio of o-phenylenediamine, copper nitrate and arginine is 1-9:3-17:2-15; Step 2): Place the mixed solution under high temperature for reaction; Step 3): Centrifuge the solution obtained in step 2), collect the upper solution, filter, dialyze, collect the solution with a molecular weight of more than 800 Da, and freeze-dry to obtain copper-doped mitochondrial targeted carbon dots; The upper solution was filtered and placed in a 800Da dialysis bag for 45-50 hours in the dark.

2. The method for preparing copper-doped mitochondria-targeted carbon dots according to claim 1, characterized in that: In step 2), the mixed solution is reacted at a high temperature of 160°C-220°C for 6-24 hours.

3. The method for preparing copper-doped mitochondria-targeted carbon dots according to claim 1, characterized in that: In step 3), the solution obtained in step 2) is centrifuged at 3000 rpm for 3-5 minutes.

4. The method for preparing copper-doped mitochondria-targeted carbon dots according to claim 1, characterized in that: In step 3), the solution in the dialysis bag after dialysis is freeze-dried at -60°C for more than 72 hours and stored away from light.

5. The copper-doped mitochondrial targeted carbon dots prepared by the method for preparing the copper-doped mitochondrial targeted carbon dots according to any one of claims 1 to 4.

6. Use of the copper-doped mitochondria-targeted carbon dots according to claim 5 in the preparation of drugs for treating brain gliomas.

7. Use of the copper-doped mitochondria-targeted carbon dots according to claim 5 in the preparation of in vivo imaging agents.

Images