Preparation method of lipid droplet-targeted fluorescent carbon quantum dot composition and weight loss application

Abstract

The application discloses a preparation method and weight-reducing application of a fluorescent carbon quantum dot composition for targeting lipid droplets. Capsaicin and o-phenylenediamine are weighed and added into anhydrous ethanol, ultrasonic dissolution is carried out to obtain a solution with a concentration of 0.02-0.06 g / ml, the solution is added into an inner container of a high-pressure reaction kettle, and the high-pressure reaction kettle is heated at 160-200 DEG C for 10-14 hours and then cooled to room temperature; after filtration, the solution is dialyzed for 46-50 hours, freeze-dried to obtain a powder, and the powder is stored at 3-5 DEG C, so as to obtain the fluorescent carbon quantum dot composition; the method has easy raw materials and simple process; the carbon quantum dot composition has good biocompatibility, has no any toxicity to animals or human bodies, can target lipid droplets in mature adipocytes and display green fluorescence; the carbon quantum dot can be applied to preparation of medicines for treating obesity diseases, and the weight-reducing effect is remarkable, and the weight of high-fat diet mice can be reduced by about 16% in three weeks; the fluorescent properties of the fluorescent carbon quantum dot itself can also play a role in monitoring the residual amount of the medicine.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a method for preparing a lipid droplet-targeting fluorescent carbon quantum dot composition and its application in weight loss. Background Technology

[0002] Obesity is one of the most serious public health problems of the 21st century. It can lead to metabolic disorders and cause a variety of health problems, such as high blood sugar and blood pressure, insulin resistance, inflammation of the body and changes in gut microbiota, and bring many complications, such as type 2 diabetes, cardiovascular disease, fatty liver and cancer.

[0003] Current treatments for obesity include dietary calorie restriction, physical exercise, the use of anti-lipogenesis drugs (such as rosiglitazone, prostaglandin E2 analogs, and L-carnitine), as well as gastric reduction surgery and liposuction. However, drug treatments have limited effectiveness and are highly toxic to the gastrointestinal tract, liver, and kidneys, while surgery carries high risks and causes significant suffering for patients. Capsaicin is a small-molecule drug with weight-loss effects, but its high toxicity and limited efficacy restrict its further application. Therefore, there is an urgent need to develop safe, effective, and non-toxic methods for treating obesity.

[0004] Carbon quantum dots (CQDs) are an emerging type of luminescent carbon nanomaterial with excellent optical properties, low or no cytotoxicity, high biocompatibility, and low synthesis cost. These properties give them unique advantages over other materials (such as small molecules) in biomedical applications such as bioimaging and drug delivery. However, research on carbon quantum dots in obesity treatment is currently limited. Therefore, it is essential to find a carbon quantum dot-based weight-loss agent (browning agent) to inhibit obesity, non-alcoholic fatty liver disease, and even diabetes. Furthermore, carbon quantum dots are non-toxic and harmless, and hold promise as a new type of polymer drug for widespread clinical application, improving the treatment efficiency for various diseases.

[0005] Although weight-loss drugs have made significant progress in recent years, many problems still need to be solved. For example, Chinese patent CN 110464775 B discloses a honeysuckle oral weight-loss drug and its preparation method. The prepared product has improved taste and can be used as a meal replacement with certain effects in preventing and treating obesity. Chinese patent CN 109865011 A discloses a weight-loss drug and its preparation method, which involves pulverizing the mixed components into powder and then manually making capsules, which has certain weight-loss and anti-rebound effects. However, existing weight-loss drugs suffer from complex steps and time-consuming processes in preparation or extraction; many weight-loss drugs are highly irritating and can cause allergies; they have significant toxic side effects, such as cardiotoxicity; many weight-loss drugs require multiple treatment courses and have poor therapeutic effects; the development process of weight-loss drugs is costly, resulting in high prices; the dosage used in treatment is relatively large; and many weight-loss drugs lack tracer capabilities. Summary of the Invention

[0006] In order to overcome the defects of the prior art, the present invention aims to provide a non-toxic, harmless, non-irritating, and highly effective fat-reducing carbon quantum dot drug composition and its preparation method, and to apply the carbon quantum dot drug composition to the treatment of obesity and the reduction of metabolic disorders; at the same time, the carbon quantum dot drug composition can be traced in vivo.

[0007] To achieve the above-mentioned objective, the present invention provides a method for preparing a lipid droplet-targeted fluorescent carbon quantum dot composition, the method comprising the following steps:

[0008] ① Solution preparation: Weigh capsaicin (Cap) and o-phenylenediamine (o-PD) and add them to anhydrous ethanol. Sonicate the solution until it is completely dissolved to obtain a solution with a mass concentration of 0.02-0.06 g / ml.

[0009] ②Preparation of carbon quantum dots: Add the solution obtained in step ① into the inner liner of a polytetrafluoroethylene high-pressure reactor, heat at 160℃~200℃ for 10~14h, and allow to cool naturally to room temperature after heating.

[0010] ③ Purification of carbon quantum dots: After filtering the solution obtained in step ③ with a 0.22μm organic nylon 66 filter, the solution was transferred to a 500 molecular weight dialysis bag and dialyzed with deionized water for 46-50 hours.

[0011] ④ Preservation of carbon quantum dots: After freezing the solution from step ④ at -18℃ to -20℃, dry it with a freeze dryer to obtain powder, which is then stored at 3℃ to 5℃; thus obtaining a fluorescent carbon quantum dot composition.

[0012] In the above technical solution, the mass ratio of capsaicin (Cap) to o-phenylenediamine (o-PD) is 1:(1-5).

[0013] Furthermore, the mass ratio of capsaicin (Cap) to o-phenylenediamine (o-PD) is 1:(1-3).

[0014] Further, in step ②, the mixture is heated at 180°C for 12 hours inside the high-pressure reactor.

[0015] Furthermore, the solution obtained in step ③ is frozen at -20°C and then dried using a freeze dryer to obtain powder, which is then stored at 4°C.

[0016] A fluorescent carbon quantum dot composition prepared by the above method exhibits green fluorescence under ultraviolet light excitation; it shows no significant toxicity to 3T3-L1 cells in the range of 0–100 μg / ml, demonstrating high biocompatibility.

[0017] The fluorescent carbon quantum dot composition exhibits intracellular lipid droplet targeting. This targeting ability was verified using the following method: First, adipocytes were induced. After 3T3-L1 cells reached confluence in 3T3-L1 complete medium, the medium was changed and cultured for another two days (contact inhibition for 2 days). Then, medium containing 3-isobutyl-1-methylxanthine IBMX (0.5 mmol / L), dexamethasone DEX (1 μmol / L), and insulin (10 μg / ml) was added and cultured for 3 days. Afterward, the medium was changed to insulin-only medium (10 μg / ml) and cultured for 4 days to obtain mature adipocytes containing a large number of lipid droplets. Next, carbon quantum dot incubation was performed. The successfully induced mature adipocytes were washed three times with phosphate-buffered saline (PBS) at pH 7.4, and then stained with Nile Red fluorescent dye and the fluorescent carbon quantum dot composition for 30 min in the dark. Background staining was removed with PBS. Finally, carbon quantum dot imaging was performed. The cell samples were observed under a fluorescence microscope (Ti2-U, Nikon, Japan) to obtain images. Nile red is a common selective hydrophobic fluorescent dye used for intracellular lipid droplets and neutral lipids. Figure 2 It is known that the fluorescent carbon quantum dot composition of this technical solution can be effectively enriched in intracellular lipid droplets and emit fluorescence. The correlation coefficient with Nile Red dye is 0.91, indicating that the fluorescent carbon quantum dot composition has good intracellular lipid droplet targeting.

[0018] The application of the above-mentioned fluorescent carbon quantum dot composition in the preparation of drugs for treating obesity was investigated. The effects of the fluorescent carbon quantum dot composition on obese mice induced by a high-fat diet (HFD) were explored experimentally. The dosage of the fluorescent carbon quantum dot composition was 6–10 mg / kg·d injected into the groin area of ​​obese mice, for 18–24 consecutive days. Changes in body weight, fat content, serum total cholesterol (TC), and triglycerides (TG) were examined in each group of mice. The experimental results ultimately showed that the fluorescent carbon quantum dot composition of this technical solution has good biocompatibility and is harmless to animals or humans; moreover, it has a significant weight loss effect, reducing the body weight of mice on a high-fat diet by approximately 16% within three weeks.

[0019] The fluorescent carbon quantum dot composition has a tracking function in vivo: the lipid droplet-targeted fluorescent carbon quantum dot composition can display the remaining amount of drug by monitoring its own fluorescence.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] The preparation method of the fluorescent carbon quantum dot composition of this invention uses readily available raw materials and involves a simple process, requiring no complex purification operations. This carbon quantum dot composition exhibits good biocompatibility and is non-toxic to animals or humans. It can target lipid droplets in mature adipocytes and displays green fluorescence. It can be applied to the preparation of drugs for treating obesity, and its significant weight loss effect has been verified, reducing the body weight of mice on a high-fat diet by 16% within three weeks. The remaining amount of drug can be observed by monitoring the fluorescence of the carbon quantum dot composition. Attached Figure Description

[0022] Figure 1 This is a flowchart illustrating the preparation and experimental process of the fluorescent carbon quantum dot composition of the present invention.

[0023] Figure 2 This is a lipid droplet targeting diagram of the carbon quantum dots in Application Example 1;

[0024] Figure 3 The graph shows the changes in body weight of mice in different drug administration groups in Example 2.

[0025] Figure 4 The graph shows the analysis results of total serum cholesterol in mice from different drug administration groups in Example 2.

[0026] Figure 5 The graph shows the analysis results of serum triglycerides in mice from different drug administration groups in Example 2;

[0027] Figure 6 The image shows the fluorescence monitoring results in mice in Example 3. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments, but this does not limit the invention in any way. To avoid redundancy, unless otherwise specified, the raw materials used in the following embodiments are all commercially available products, and the methods used are all conventional methods unless otherwise specified.

[0029] A method for preparing a lipid droplet-targeted fluorescent carbon quantum dot composition, such as... Figure 1 As shown, it includes the following steps:

[0030] ① Solution preparation: Weigh capsaicin and o-phenylenediamine and add them to anhydrous ethanol. Sonicate until completely dissolved to obtain a solution with a mass concentration of 0.02-0.06 g / ml.

[0031] ②Preparation of carbon quantum dots: Add the solution obtained in step ① into the inner liner of a polytetrafluoroethylene high-pressure reactor, heat at 160℃~200℃ for 10~14h, and allow to cool naturally to room temperature after heating is complete;

[0032] ③ Purification of carbon quantum dots: After filtering the solution obtained in step ② with a 0.22μm organic nylon 66 filter, the solution was transferred to a 500 molecular weight dialysis bag and dialyzed with deionized water for 46-50 hours.

[0033] ④ Preservation of carbon quantum dots: After freezing the solution obtained in step ③ at -18℃ to -22℃, dry it with a freeze dryer to obtain powder, which is then stored at 3℃ to 5℃; thus obtaining a fluorescent carbon quantum dot composition.

[0034] Any aspects not described in the following embodiments are the same as those described in the specific embodiments above.

[0035] In the specific implementation, capsaicin (Cap) was provided by Shanghai Maclean Biochemical Technology Co., Ltd.; o-phenylenediamine (o-PD) was provided by Shanghai Aladdin Biochemical Technology Co., Ltd.; anhydrous ethanol was provided by Tianjin Fuyu Fine Chemical Co., Ltd.; 3T3-L1 cells and 3T3-L1 complete culture medium were provided by the Shanghai Cell Bank of the Chinese Academy of Sciences (Cell Resource Center of the Institute of Cell Biology and Cell Engineering, Shanghai Institute of Life Sciences, Chinese Academy of Sciences); dexamethasone (DEX) was provided by Merck AG, Germany; and animals (healthy male C57BL6 / J mice, six weeks old) were provided by Beijing Huafukang Co., Ltd.

[0036] Example 1

[0037] A method for preparing a lipid droplet-targeted fluorescent carbon quantum dot composition includes the following steps:

[0038] ① Solution preparation: Weigh 0.1g capsaicin Cap and 0.3g o-phenylenediamine o-PD and add them to 10ml anhydrous ethanol. Sonicate to dissolve them completely to obtain a solution with a mass concentration of 0.04g / ml.

[0039] ②Preparation of carbon quantum dots: The solution obtained in step ① is added to the inner liner of a polytetrafluoroethylene high-pressure reactor and heated at 180°C for 12 hours. After heating, it is allowed to cool naturally to room temperature.

[0040] ③ Purification of carbon quantum dots: After filtering the solution obtained in step ② with a 0.22μm organic nylon 66 filter, the solution was transferred to a 500 molecular weight dialysis bag and dialyzed with deionized water for 48 hours.

[0041] ④ Preservation of carbon quantum dots: After freezing the solution obtained in step ③ at -20℃, dry it with a freeze dryer to obtain powder, which is then stored at 4℃; thus, a fluorescent carbon quantum dot composition is obtained.

[0042] Comparative Example 1

[0043] A method for preparing a lipid droplet-targeted fluorescent carbon quantum dot composition includes the following steps:

[0044] ① Solution preparation: Weigh 0.3g capsaicin Cap and 0g o-phenylenediamine o-PD and add them to 10ml anhydrous ethanol. Sonicate to dissolve them completely to obtain a solution with a mass concentration of 0.04g / ml.

[0045] ②Preparation of carbon quantum dots: The solution obtained in step ① was added to the inner liner of a polytetrafluoroethylene high-pressure reactor and heated at 180°C for 12 hours. After heating, it was naturally cooled to room temperature. It was found that fluorescent carbon quantum dot compositions could not be obtained.

[0046] Comparative Example 2

[0047] A method for preparing a lipid droplet-targeted fluorescent carbon quantum dot composition includes the following steps:

[0048] ① Solution preparation: Weigh 0.3g capsaicin Cap and 0.1g o-phenylenediamine o-PD and add them to 10ml anhydrous ethanol. Sonicate to dissolve them completely to obtain a solution with a mass concentration of 0.04g / ml.

[0049] ②Preparation of carbon quantum dots: The solution obtained in step ① was added to the inner liner of a polytetrafluoroethylene high-pressure reactor and heated at 180°C for 12 hours. After heating, it was naturally cooled to room temperature. It was found that fluorescent carbon quantum dot compositions could not be obtained.

[0050] Comparative Example 3

[0051] A method for preparing a lipid droplet-targeted fluorescent carbon quantum dot composition includes the following steps:

[0052] ① Solution preparation: Weigh 0.1g capsaicin Cap and 0.3g o-phenylenediamine o-PD and add them to 10ml of water. Sonicate until completely dissolved to obtain a solution with a mass concentration of 0.04g / ml.

[0053] Preparation of carbon quantum dots: The solution obtained in step ① was added to the inner liner of a polytetrafluoroethylene high-pressure reactor and heated at 180°C for 12 hours. After heating, it was naturally cooled to room temperature. It was found that fluorescent carbon quantum dot compositions could not be obtained.

[0054] Comparative Example 4

[0055] A method for preparing a lipid droplet-targeted fluorescent carbon quantum dot composition includes the following steps:

[0056] ① Solution preparation: Weigh 0.1g capsaicin Cap and 0.3g o-phenylenediamine o-PD and add them to 10ml anhydrous ethanol. Sonicate to dissolve them completely to obtain a solution with a mass concentration of 0.04g / ml.

[0057] ②Preparation of carbon quantum dots: The solution obtained in step ① was added to the inner liner of a polytetrafluoroethylene high-pressure reactor and heated at 120°C for 12 hours. After heating, it was naturally cooled to room temperature. It was found that fluorescent carbon quantum dot compositions could not be obtained.

[0058] Application Example 1

[0059] A method for preparing a lipid droplet-targeting fluorescent carbon quantum dot composition, wherein the fluorescent carbon quantum dot composition has intracellular lipid droplet targeting properties.

[0060] The intracellular lipid droplet targeting ability of the fluorescent carbon quantum dot composition was verified by the following method:

[0061] ① Induction of adipocytes: After 3T3-L1 cells have grown to confluence in 3T3-L1 complete medium, the medium is changed and cultured for another two days (contact inhibition for 2 days); then, medium containing 3-isobutyl-1-methylxanthine IBMX (concentration 0.5 mmol / L), dexamethasone DEX (1 μmol / L) and insulin (10 μg / ml) is added and cultured for 3 days. After that, the medium is changed to insulin (10 μg / ml) only and cultured for 4 days to obtain mature adipocytes containing a large number of lipid droplets.

[0062] ② Incubation of carbon quantum dots: The mature adipocytes that were successfully induced were washed three times with PBS (pH 7.4), and then stained with Nile Red fluorescent dye and the fluorescent carbon quantum dot composition prepared in Example 1 for 30 min in the dark; the background staining was removed with PBS.

[0063] ③ Imaging of carbon quantum dots: The above cell samples were observed under a fluorescence microscope (Ti2-U, Nikon, Japan) to obtain images, as shown in the attached figure. Figure 2 As shown; Nile red is a common selective hydrophobic fluorescent dye used for intracellular lipid droplets and neutral lipids, produced by... Figure 2It can be seen that the fluorescent carbon quantum dot composition of this technical solution can be effectively enriched in intracellular lipid droplets and emit fluorescence, with a superposition correlation coefficient of 0.91, indicating that it has good intracellular lipid droplet targeting.

[0064] Application Example 2

[0065] The use of the lipid droplet-targeting fluorescent carbon quantum dot composition in the preparation of a drug for treating obesity.

[0066] The effects of the fluorescent carbon quantum dot composition prepared in Example 1 on high-fat diet (HFD) induced obesity in mice were investigated by experiment. Thirty mice were randomly divided into a control group, a capsaicin group, and the carbon quantum dot group of the present invention.

[0067] All groups (control group, capsaicin group, and carbon quantum dot group) were given a high-fat diet to induce an obesity model in mice for four weeks. After that, in addition to being fed a high-fat diet every day, the mice in each group were given phosphate-buffered saline (PBS) injections in the groin of the control group; the capsaicin group was injected with capsaicin Cap at 8 mg / kg·d in the groin of the mice; and the carbon quantum dot group was injected with the carbon quantum dot composition prepared in Example 1 at 8 mg / kg·d in the groin of the obese mice. All three groups were administered the drugs continuously for 21 days. After the last administration, the mice were fasted for 12 hours and then euthanized by cervical dislocation.

[0068] The changes in body weight, fat content, serum total cholesterol (TC), and triglycerides (TG) were examined in each group of mice. Figure 3 The results of body weight changes in mice in different drug administration groups were obtained from... Figure 3 It can be seen that, under conditions of a consistently high-fat diet, the untreated group's weight increased to 108.4% of its original weight. Both the capsaicin group and the carbon quantum dot group of the present invention showed weight reduction effects, with the capsaicin group reducing weight to 99.9% of its original weight and the carbon quantum dot group reducing weight to 92% of its original weight. The carbon quantum dot group of the present invention exhibited a better therapeutic effect than capsaicin alone; this demonstrates that the carbon quantum dot composition weight-loss drug prepared using the technical solution of the present invention has a very good effect on weight reduction.

[0069] The changes in serum total cholesterol (TC) levels in each group of mice are as follows: Figure 4 As shown, by Figure 4 It can be seen that, under the condition of a consistently high-fat diet, both the capsaicin group and the present invention group (carbon quantum dot group) have the effect of reducing serum cholesterol. Compared with the control group, the capsaicin group reduced triglycerides to 82.2% and the present invention group (carbon quantum dot group) reduced total cholesterol to 71.7%. Therefore, the present invention group (carbon quantum dot group) showed a better therapeutic effect than capsaicin alone.

[0070] The changes in serum triglyceride (TG) levels in each group of mice are as follows: Figure 5 As shown, by Figure 5It can be seen that, under the condition of a consistently high-fat diet, both the capsaicin group and the present invention group (carbon quantum dot group) have the effect of reducing serum triglycerides. Compared with the control group, the triglycerides in the capsaicin group were reduced to 70.6%, and the triglycerides in the present invention group (carbon quantum dot group) were reduced to 47.6%. Therefore, the present invention group showed a better therapeutic effect than capsaicin alone.

[0071] Application Example 3

[0072] Application of the lipid droplet-targeting fluorescent carbon quantum dot composition in the preparation of drugs for treating obesity: The lipid droplet-targeting fluorescent carbon quantum dot composition can display the remaining amount of drug by monitoring its own fluorescence, that is, the fluorescent carbon quantum dot composition has a tracer function in vivo.

[0073] The fluorescence characteristics of the fluorescent carbon quantum dot composition prepared in Example 1 were investigated in vivo through experiments: Three mice were injected with the carbon quantum dot group of the present invention in the groin area, and the fluorescence of the carbon quantum dot group was monitored using a small animal imaging system to monitor the remaining amount of the drug. The in vivo fluorescence images of the mice at different time points are shown below. Figure 6 As shown, by Figure 6 It is known that the present invention group (carbon quantum dot group) emits strong fluorescence in the abdomen of mice, and the fluorescence shows that the carbon quantum dots will be completely metabolized after 36 hours.

[0074] The above application examples demonstrate that the fluorescent carbon quantum dot composition of this technical solution has good biocompatibility and is harmless to animals or humans; moreover, it has a significant weight loss effect, reducing the weight of mice on a high-fat diet by about 16% within three weeks.

[0075] For anyone skilled in the art, many possible variations and modifications can be made to the technical solutions of this invention, or equivalent embodiments can be modified based on the disclosed technical content, without departing from the scope of the technical solutions of this invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this invention without departing from the content of the technical solutions of this invention should still fall within the protection scope of the technical solutions of this invention.

Claims

1. A method for preparing a lipid droplet-targeted fluorescent carbon quantum dot composition, characterized in that, The method includes the following steps: ① Solution preparation: Weigh 0.1 g capsaicin and 0.3 g o-phenylenediamine and add them to 10 ml of anhydrous ethanol. Sonicate until completely dissolved to obtain a solution with a mass concentration of 0.04 g / ml. ②Preparation of carbon quantum dots: The solution obtained in step ① was added to the inner liner of a polytetrafluoroethylene high-pressure reactor and heated at 180°C for 12 h. After heating, it was allowed to cool naturally to room temperature. ③ Purification of carbon quantum dots: After filtering the solution obtained in step ② with a 0.22 μm organic nylon 66 filter, the solution was transferred to a 500 molecular weight dialysis bag and dialyzed with deionized water for 46-50 h. ④ Preservation of carbon quantum dots: After freezing the solution obtained in step ③ at -20℃, dry it with a freeze dryer to obtain powder, which is then stored at 4℃; thus, a fluorescent carbon quantum dot composition is obtained.

2. A fluorescent carbon quantum dot composition prepared by the method according to claim 1.

3. The fluorescent carbon quantum dot composition according to claim 2, characterized in that, The fluorescent carbon quantum dot composition exhibits intracellular lipid droplet targeting.

4. The use of the fluorescent carbon quantum dot composition as described in claim 2 or 3 in the preparation of a medicament for treating obesity.

5. The application according to claim 4, characterized in that, The dosage of the fluorescent carbon quantum dot composition is 6-10 mg / kg·d of carbon quantum dots injected into the groin of mice, for 18-24 consecutive days.

6. The application according to claim 5, characterized in that, The fluorescent carbon quantum dot composition has a tracer function in vivo.

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