Drug-loaded nano carbon dot for treating atherosclerosis and preparation method thereof

Abstract

The invention discloses a drug-loaded nano carbon dot for treating atherosclerosis and a preparation method of the drug-loaded nano carbon dot, and belongs to the technical field of biological medicines. Malic acid, vitamin B1 and histamine phosphate are used as precursors, a one-step hydrothermal method is adopted to synthesize nitrogen-sulfur-phosphorus co-doped nano carbon dots NSP-CDs, a monascus extract and serotonin are loaded on the surfaces of the carbon dots through electrostatic adsorption, and the nano drug which is simple in structure and small in size and has a targeted therapy effect is prepared. The nano-drug has low cytotoxicity and good in-vivo biological safety, and can obviously reduce total cholesterol, triglyceride, low-density lipoprotein cholesterol and Hamp of a sick mouse; the results of E staining and oil red O staining show that the plaque condition of the atherosclerosis model mouse after treatment is obviously improved. The monascus extract loaded nano-drug provided by the invention can be used for treating atherosclerosis diseases, and is expected to become a novel drug for preventing and treating atherosclerosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to drug-loaded nano-carbon dots for treating atherosclerosis and a preparation method thereof. Background Art

[0002] Atherosclerosis (AS) is the basic pathology of cardiovascular diseases worldwide, which can lead to myocardial infarction, angina pectoris, ischemic heart disease, ischemic stroke, hypertension, coronary heart disease, stroke and other diseases, seriously threatening human health. Therefore, preventing the occurrence and progression of AS has become the research focus of cardiovascular disease prevention and treatment. Inflammation is an important driving force in the occurrence and development of AS. More and more studies have shown that inflammatory factors and inflammasomes play a key role in the inflammatory response of AS.

[0003] Commonly used drugs for atherosclerosis include lipid-lowering drugs, and statins are the first choice for lipid-lowering drugs. However, long-term use of statins may cause adverse reactions such as liver and kidney damage, gastrointestinal reactions, and myopathy.

[0004] Red yeast rice (HQ) is made from the mycelium of the Aspergillus fungus Monascus purpureus Went. parasitizing on japonica rice. It has the effects of strengthening the spleen and digestion, promoting blood circulation and removing blood stasis. Nanocarbon quantum dots (abbreviated as: carbon dots; CDs) are a new type of carbon nanomaterial with a particle size of less than 10nm and fluorescent properties. They have excellent optical properties and medical properties and are widely used in cell imaging, biosensors, disease diagnosis, drug carriers and other fields. Summary of the invention

[0005] In view of the above-mentioned prior art, the present invention provides a drug-loaded nano-carbon dot for treating atherosclerosis and a preparation method thereof, so as to solve the technical problems that the existing drugs for treating atherosclerosis have poor therapeutic effects and strong side effects.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is to provide a method for preparing drug-loaded nanocarbon dots for treating atherosclerosis, comprising the following steps:

[0007] S1: mixing malic acid, vitamin B1 and histamine phosphate, and dissolving the mixture in a solvent to obtain a mixed solution;

[0008] S2: The mixed solution is reacted at 140-240°C for 1-12 hours, then filtered, the filtrate is put into a dialysis bag, and dialyzed with pure water for 24 hours to obtain a carbon dot solution; the carbon dot solution is then freeze-dried to obtain nano-carbon dots;

[0009] S3: extracting red yeast rice with an ethanol solution of sodium hydroxide to obtain a red yeast rice extract;

[0010] S4: dissolving the red yeast rice extract in dilute hydrochloric acid to prepare a solution A with a concentration of 10 mg / ml; dissolving the nanocarbon dots in PBS solution to prepare a solution B with a concentration of 10 mg / ml;

[0011] S5: Mix solution A and solution B, and then stir and react at room temperature in the dark for 20 to 24 hours, and then dialyze the reaction solution; then add 5-HT to the dialyzed reaction solution, continue to stir and react at room temperature in the dark for 20 to 24 hours, and then dialyze and freeze-dry to obtain.

[0012] Based on the above technical solution, the present invention can also be improved as follows.

[0013] Furthermore, the mass ratio of malic acid, vitamin B1 and histamine phosphate is 1-3:1-3:1-3.

[0014] Furthermore, the solvent is water, and the solid-liquid ratio of the mixture to water is 0.7 g:20 ml.

[0015] Furthermore, the filtration in S2 is performed using a 0.22 μm syringe filter; the molecular weight cutoff of the dialysis bag is 1000 Da.

[0016] Further, the red yeast rice extract is prepared by the following steps:

[0017] Immerse the red yeast rice in an ethanol solution of sodium hydroxide and reflux for 2 to 3 times, each time for 1 to 2 hours; then combine the filtrates and concentrate under reduced pressure until the volume no longer changes.

[0018] Further, the ethanol solution of sodium hydroxide is obtained by dissolving sodium hydroxide in 75% ethanol, and the concentration of sodium hydroxide in the solution is 0.1 mol / L.

[0019] Furthermore, the concentration of the PBS solution was 0.01 M and the pH was 7.

[0020] Furthermore, the volume ratio of solution A to solution B is 1:1.5-3.

[0021] Furthermore, the mass ratio of 5-HT to nano-carbon dots is 1:0.75-1.5.

[0022] The invention also discloses drug-loaded nano carbon dots for treating atherosclerosis, which are prepared by the preparation method.

[0023] The beneficial effects of the present invention are:

[0024] The present invention uses malic acid, vitamin B1, and histamine phosphate as precursors, and adopts a one-step hydrothermal method to synthesize nitrogen, sulfur, and phosphorus co-doped nanocarbon dots NSP-CDs. The surface functional groups of the nanocarbon dots are rich, including hydroxyl, carboxyl, amino, and heterocyclic groups; red yeast rice extract and 5-hydroxytryptamine (5-HT) are then loaded on the surface of the carbon dots by electrostatic adsorption to obtain a nanomedicine NSP-CDs@HQ@5-HT with a simple structure, small size, and better therapeutic effect. NSP-CDs@HQ@5-HT is used for ApoE - / - The treatment study of atherosclerosis model mice showed that the NSP-CDs@HQ@5-HT treatment group could significantly reduce the total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) of the diseased mice compared with the red yeast extract treatment group; H&E staining and Oil Red O staining results showed that the plaque condition of the atherosclerosis model mice improved significantly after treatment, and the immunohistochemical analysis results also showed that compared with the blank control group and the red yeast extract treatment group, the CD68 and MCP-1 expressions of the targeted nanomedicine group mice were lower. The tissue section H&E chromosome intrachromosomal toxicity assessment experiment showed that the nanomedicine has good biosafety and can be used for the treatment of atherosclerosis, providing the possibility for its further clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 TEM image of nanocarbon dots (NPS-CDs);

[0026] Figure 2 is the UV spectrum of nanocarbon dots (NPS-CDs);

[0027] Figure 3 IR spectrum of nanocarbon dots (NPS-CDs);

[0028] Figure 4 Zeta potential diagram of NSP-CDs@HQ;

[0029] Figure 5 Zeta potential diagram of NSP-CDs@HQ@5-HT;

[0030] Figure 6 TEM image of NSP-CDs@HQ;

[0031] Figure 7 TEM image of NSP-CDs@HQ@5-HT;

[0032] Figure 8 This is the safety evaluation result diagram of NSP-CDs@HQ@5-HT;

[0033] Fig. 9NSP-CDs@HQ@5-H for ApoE - / - Graph showing the therapeutic effects of AS mice. DETAILED DESCRIPTION

[0034] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0035] Example 1

[0036] A drug-loaded nanocarbon dot for treating atherosclerosis is prepared by the following steps:

[0037] S1: malic acid, vitamin B1 and histamine phosphate are mixed in a mass ratio of 1:3:3, and the mixture is dissolved in ultrapure water in a material-liquid ratio of 0.7 g:20 ml to obtain a mixed solution;

[0038] S2: The mixed solution was transferred to a 25 ml polytetrafluoroethylene liner, and then the polytetrafluoroethylene liner was turned over into the reactor, and the reaction was carried out at 200°C for 8 hours, and then the reaction solution was filtered with a 0.22 μm syringe filter, and the filtrate was placed in a dialysis bag with a molecular weight cutoff of 1000 Da, and dialyzed with pure water for 24 hours to remove unreacted small molecules, etc., to obtain a carbon dot solution; and then the carbon dot solution was freeze-dried with a freeze dryer to obtain nanocarbon dots (NPS-CDs);

[0039] S3: Red yeast rice (purchased from Zhejiang Sanhe Biotechnology Co., Ltd.) was immersed in an ethanol solution of sodium hydroxide and refluxed for extraction 3 times, each time for 1 hour; then the filtrates were combined and concentrated under reduced pressure until the volume no longer changed, to obtain a red yeast rice extract; the ethanol solution of sodium hydroxide used was obtained by dissolving sodium hydroxide in 75% ethanol, and the concentration of sodium hydroxide in the solution was 0.1 mol / L;

[0040] S4: dissolving the red yeast rice extract in dilute hydrochloric acid to prepare a solution A with a concentration of 10 mg / ml; dissolving NPS-CDs in PBS solution (0.01 M, pH = 7.0) to prepare a solution B with a concentration of 10 mg / ml;

[0041] S5: Solution A and solution B were mixed in a volume ratio of 1:2, and then stirred in the dark at room temperature for 24 hours, and then the reaction solution was dialyzed with a dialysis bag with a molecular weight cutoff of 1000Da for 24 hours (pure water dialysis) to obtain NPS-CDs@HQ solution; then 5-HT (5-hydroxytryptamine) was added to the NPS-CDs@HQ solution, and the mass ratio of the added 5-HT to NPS-CDs was 1:1, and the reaction was continued in the dark at room temperature with stirring for 24 hours, and then the reaction solution was dialyzed with a dialysis bag with a molecular weight cutoff of 1000Da for 24 hours (pure water dialysis), and then freeze-dried with a freeze dryer to obtain drug-loaded nanocarbon dots NPS-CDs@HQ@5-HT for the treatment of atherosclerosis.

[0042] Example 2

[0043] A drug-loaded nanocarbon dot for treating atherosclerosis is prepared by the following steps:

[0044] S1: malic acid, vitamin B1 and histamine phosphate are mixed in a mass ratio of 3:1:3, and the mixture is dissolved in ultrapure water in a material-liquid ratio of 0.7 g:20 ml to obtain a mixed solution;

[0045] S2: The mixed solution was transferred to a 25 ml polytetrafluoroethylene liner, and then the polytetrafluoroethylene liner was turned over into the reactor, and the reaction was carried out at 140°C for 12 hours, and then the reaction solution was filtered with a 0.22 μm syringe filter, and the filtrate was placed in a dialysis bag with a molecular weight cutoff of 1000 Da, and dialyzed with pure water for 24 hours to remove unreacted small molecules, etc., to obtain a carbon dot solution; and then the carbon dot solution was freeze-dried with a freeze dryer to obtain nanocarbon dots (NPS-CDs);

[0046] S3: Red yeast rice (purchased from Zhejiang Sanhe Biotechnology Co., Ltd.) was immersed in an ethanol solution of sodium hydroxide, and refluxed for extraction twice, each time for 2 hours; then the filtrates were combined and concentrated under reduced pressure until the volume no longer changed, to obtain a red yeast rice extract; the ethanol solution of sodium hydroxide used was obtained by dissolving sodium hydroxide in 75% ethanol, and the concentration of sodium hydroxide in the solution was 0.1 mol / L;

[0047] S4: dissolving the red yeast rice extract in dilute hydrochloric acid to prepare a solution A with a concentration of 10 mg / ml; dissolving NPS-CDs in PBS solution (0.01 M, pH = 7.0) to prepare a solution B with a concentration of 10 mg / ml;

[0048] S5: Solution A and solution B were mixed in a volume ratio of 1:1.5, and then stirred in the dark at room temperature for 20 hours, and then the reaction solution was dialyzed with a dialysis bag with a molecular weight cutoff of 1000Da for 24 hours (pure water dialysis) to obtain NPS-CDs@HQ solution; then 5-HT (5-hydroxytryptamine) was added to the NPS-CDs@HQ solution, and the mass ratio of the added 5-HT to NPS-CDs was 1:0.75, and the reaction was continued in the dark at room temperature with stirring for 24 hours, and then the reaction solution was dialyzed with a dialysis bag with a molecular weight cutoff of 1000Da for 24 hours (pure water dialysis), and then freeze-dried with a freeze dryer to obtain drug-loaded nanocarbon dots NPS-CDs@HQ@5-HT for the treatment of atherosclerosis.

[0049] Example 3

[0050] A drug-loaded nanocarbon dot for treating atherosclerosis is prepared by the following steps:

[0051] S1: malic acid, vitamin B1 and histamine phosphate are mixed in a mass ratio of 3:3:1, and the mixture is dissolved in ultrapure water in a material-liquid ratio of 0.7 g:20 ml to obtain a mixed solution;

[0052] S2: The mixed solution was transferred to a 25 ml polytetrafluoroethylene liner, and then the polytetrafluoroethylene liner was turned over into the reactor, and the reaction was carried out at 240°C for 1 hour, and then the reaction solution was filtered with a 0.22 μm syringe filter, and the filtrate was placed in a dialysis bag with a molecular weight cutoff of 1000 Da, and dialyzed with pure water for 24 hours to remove unreacted small molecules, etc., to obtain a carbon dot solution; and then the carbon dot solution was freeze-dried with a freeze dryer to obtain nanocarbon dots (NPS-CDs);

[0053] S3: Red yeast rice (purchased from Zhejiang Sanhe Biotechnology Co., Ltd.) was immersed in an ethanol solution of sodium hydroxide and refluxed for extraction 3 times, each time for 1 hour; then the filtrates were combined and concentrated under reduced pressure until the volume no longer changed, to obtain a red yeast rice extract; the ethanol solution of sodium hydroxide used was obtained by dissolving sodium hydroxide in 75% ethanol, and the concentration of sodium hydroxide in the solution was 0.1 mol / L;

[0054] S4: dissolving the red yeast rice extract in dilute hydrochloric acid to prepare a solution A with a concentration of 10 mg / ml; dissolving NPS-CDs in PBS solution (0.01 M, pH = 7.0) to prepare a solution B with a concentration of 10 mg / ml;

[0055] S5: Solution A and solution B were mixed in a volume ratio of 1:3, and then stirred in the dark at room temperature for 24 hours, and then the reaction solution was dialyzed with a dialysis bag with a molecular weight cutoff of 1000Da for 24 hours (pure water dialysis) to obtain NPS-CDs@HQ solution; then 5-HT (5-hydroxytryptamine) was added to the NPS-CDs@HQ solution, and the mass ratio of the added 5-HT to NPS-CDs was 1:1.5, and the reaction was continued in the dark at room temperature for 20 hours, and then the reaction solution was dialyzed with a dialysis bag with a molecular weight cutoff of 1000Da for 24 hours (pure water dialysis), and then freeze-dried with a freeze dryer to obtain drug-loaded nanocarbon dots NPS-CDs@HQ@5-HT for the treatment of atherosclerosis.

[0056] Experimental example

[0057] The drug-loaded nano-carbon dots for treating atherosclerosis prepared in the above Examples 1 to 3 have similar performances. Taking the drug-loaded nano-carbon dots for treating atherosclerosis prepared in Example 1 as an example, its performance is described in detail.

[0058] 1. Structural Characterization of NPS-CDs

[0059] The structure of the nano-carbon dots (NPS-CDs) prepared in Example 1S2 was characterized by transmission electron microscopy. Figure 1As shown (the inset figure is the particle size distribution diagram of NPS-CDs). It can be seen from the figure that the nano carbon dots are a type of spherical nanoparticles that are evenly dispersed in the solution and have a particle size of less than 10 nm (mainly distributed in 4-8 nm).

[0060] The nano carbon dots (NPS-CDs) prepared in Example 1S2 were measured by UV. The results are as follows: Figure 2 As shown in the figure, NPS-CDs has absorption at 250nm, indicating that the structure contains conjugated systems such as benzene rings.

[0061] The nano carbon dots (NPS-CDs) prepared in Example 1S2 were subjected to IR measurement, and the results were as follows: Figure 3 As shown in the figure, NPS-CDs is between 3000 and 2500 cm -1 There is a broad peak, which is the -OH and -NH 2 Stretching vibration peak; 1696cm -1 The strong absorption peak corresponds to the stretching vibration peak of C=O, which comes from -COOH in malic acid. It can be seen that the surface functional groups of NPS-CDs are rich, including hydroxyl, carboxyl, amino and other active functional groups, which is conducive to subsequent structural modification.

[0062] 2. Structural Characterization of NPS-CDs@HQ@5-HT

[0063] The potential values ​​of NPS-CDs@HQ and NPS-CDs@HQ@5-HT were measured by Zeta potential instrument, and the morphology of NPS-CDs@HQ and NPS-CDs@HQ@5-HT was tested by transmission electron microscopy (TEM) and the particle size was calculated. The potential test results of NPS-CDs@HQ and NPS-CDs@HQ@5-HT are shown in Figure 2. Figure 4 and Figure 5 As shown in the figure, after NPS-CDs was coupled with negatively charged red yeast rice extract, the potential of NPS-CDs@HQ changed to -2.96mv ( Figure 4 ), with the coupling of positively charged 5-HT, the potential of NPS-CDs@HQ@5-HT further changed to -0.222mv( Figure 5 ), indicating that the coupling of red yeast rice extract and 5-HT changed the electrical properties and potential values ​​of carbon dots, proving that the drug and targeting substrate were successfully coupled to the surface of carbon dots.

[0064] TEM test results of NPS-CDs@HQ and NPS-CDs@HQ@5-HT are shown in Figure 6 and 7 As shown in the figure, it can be seen that NPS-CDs@HQ is a transparent round shape, and the particle size increases to about 30nm ( Figure 6), NPS-CDs@HQ@5-HT became a tightly connected network structure, and the particle size changed to 61nm ( Figure 7 ), indicating that the surface of carbon dots has been successfully modified.

[0065] 3. Biosafety of NPS-CDs@HQ@5-HT

[0066] After mice were injected with NPS-CDs@HQ@5-HT via the tail vein, the heart, liver, spleen, lung, and kidney of the mice were carefully separated and H&E staining was performed on these organs. The results are shown in the figure. Figure 8 As shown in the figure, compared with the saline control group, after injection of the nanodrug NPS-CDs@HQ@5-HT, the main tissues and organs of the mice did not show obvious morphological changes, and no obvious pathological damage was observed, proving that NPS-CDs@HQ@5-HT has good biocompatibility and low toxicity in treatment.

[0067] IV. Study on the treatment of atherosclerotic plaques in mice by NPS-CDs@HQ@5-HT

[0068] 1. ApoE - / - Construction of atherosclerotic plaque model in transgenic mice

[0069] Select 5-week-old male ApoE - / - Transgenic mice, weighing 15-20g, a total of 25 mice, Beijing Huafukang Experimental Animal Center, experimental animal license number: SCXK (Beijing) 2019-0008. All mice were kept in a specific pathogen free SPF (specific pathogen free) animal room, the breeding environment temperature was maintained at 20-25°C, the relative humidity was maintained at 50%-65%, and they were fed with Western diet for 20 consecutive weeks to establish an atherosclerosis model. The body weight of mice, blood biochemical indicators (TC, TG, LDL-C, HDL-C), and aortic tissue pathology sections were measured to comprehensively evaluate ApoE - / - The mouse AS plaque model was successfully established.

[0070] 2. Effect of NPS-CDs@HQ@5-HT on ApoE - / - Treatment outcomes in AS mice

[0071] Take the ApoE model successfully - / -Mice were randomly divided into 3 groups, 3 mice in each group: NPS-CDs@HQ@5-HT nanomedicine treatment group, red yeast rice extract treatment group and blank control group. The NPS-CDs@HQ@5-HT group was injected with nanomedicine NPS-CDs@HQ@5-HT equivalent to 10 mg / kg of red yeast rice extract through the tail vein, the red yeast rice extract treatment group was injected with 10 mg / kg of red yeast rice extract through the tail vein, and the control group was injected with an equal volume of normal saline. After 1 month of treatment, the normal blood lipid values ​​of mice in each group were measured by an automatic biochemical analyzer, and the results are shown in Table 1.

[0072] Table 1 Blood lipid levels of ApoE- / -AS mice in different experimental groups (mean ± SD, n = 3)

[0073]

[0074] As can be seen from Table 1, after treatment with NPS-CDs@HQ@5-HT and red yeast rice extract, the TC, TG, and LDL-C of mice decreased relative to the control group, and the HDL-C concentration increased. By comparison, the TC, TG, and LDL-C values ​​of the NPS-CDs@HQ@5-HT group decreased, and the HDL-C value increased more significantly, about 1.5 to 2 times the change in the red yeast rice extract group, indicating that the treatment effect of the NPS-CDs@HQ@5-HT group was better, confirming the targeted therapeutic effect of the probe. To further evaluate the improvement of aortic plaques in mice, the aortas of each group of mice were sectioned and analyzed by H&E staining and Oil Red O staining. The results are shown in the figure. Fig. 9 As shown in the figure, it can be seen that the treatment with red yeast rice nanomedicine (NPS-CDs@HQ@5-HT) significantly improved the condition of plaques. The results of immunohistochemical analysis also showed that the expression of CD68 and MCP-1 in mice in the nanomedicine group was lower than that in the control group and red yeast rice extract group.

[0075] In summary, the results of blood lipid data, section staining, and immunohistochemical analysis all indicate that the nanodrug NPS-CDs@HQ@5-HT has a targeted therapeutic effect on AS mice.

[0076] Although the specific implementation of the present invention is described in detail in conjunction with the embodiments, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A method for preparing drug-loaded nanocarbon dots for treating atherosclerosis, characterized in that: The following steps are involved: S1: mixing malic acid, vitamin B1 and histamine phosphate, and dissolving the mixture in a solvent to obtain a mixed solution; S2: The mixed solution is reacted at 140-240°C for 1-12 hours, then filtered, the filtrate is put into a dialysis bag, and dialyzed with pure water for 24 hours to obtain a carbon dot solution; the carbon dot solution is then freeze-dried to obtain nano-carbon dots; S3: extracting red yeast rice with an ethanol solution of sodium hydroxide to obtain a red yeast rice extract; S4: dissolving the red yeast rice extract in dilute hydrochloric acid to prepare a solution A with a concentration of 10 mg / ml; dissolving the nanocarbon dots in PBS solution to prepare a solution B with a concentration of 10 mg / ml; S5: Mix solution A and solution B, and then stir and react at room temperature in the dark for 20 to 24 hours, and then dialyze the reaction solution; then add 5-HT to the dialyzed reaction solution, continue to stir and react at room temperature in the dark for 20 to 24 hours, and then dialyze and freeze-dry to obtain.

2. The preparation method according to claim 1, characterized in that: The mass ratio of the malic acid, vitamin B1 and histamine phosphate is 1-3:1-3:1-3.

3. The preparation method according to claim 2, characterized in that: The solvent is water, and the solid-liquid ratio of the mixture to water is 0.7g:20ml.

4. The preparation method according to claim 1, characterized in that: The filtration in S2 is performed using a 0.22 μm syringe filter; the molecular weight cutoff of the dialysis bag is 1000 Da.

5. The preparation method according to claim 1, characterized in that: The red yeast rice extract is prepared by the following steps: Immerse the red yeast rice in an ethanol solution of sodium hydroxide and reflux for 2 to 3 times, each time for 1 to 2 hours; then combine the filtrates and concentrate under reduced pressure until the volume no longer changes.

6. The preparation method according to claim 4, characterized in that: The sodium hydroxide ethanol solution is obtained by dissolving sodium hydroxide in 75% ethanol, and the concentration of sodium hydroxide in the solution is 0.1 mol / L.

7. The preparation method according to claim 1, characterized in that: The concentration of the PBS solution is 0.01 M and the pH is 7.

8. The preparation method according to claim 1, characterized in that: The volume ratio of solution A to solution B is 1:1.5-3.

9. The preparation method according to claim 8, characterized in that: The mass ratio of the 5-HT to the nano-carbon dots is 1:0.75-1.

5.

10. Drug-loaded nano-carbon dots for treating atherosclerosis obtained by the preparation method according to any one of claims 1 to 9.

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