Carbon nanodot composite amino acid reagent, preparation method and application thereof

Abstract

The application discloses carbon nanodot composite amino acid reagents, a preparation method and application thereof, and belongs to the field of biological materials. The carbon nanodot composite amino acid reagent comprises carbon nanodots and amino acids which are compounded with each other, and the carbon nanodots have electron-withdrawing groups on the surface. The carbon nanodots with electron-withdrawing groups on the surface and the amino acids are compounded, and the two are combined together through hydrogen bonds to form carbon nanodot amino acid reagents. The carbon nanodot amino acid reagents have the properties of amino acids and carbon dots without a complicated purification procedure, are easy to combine with cell membranes to pass through the cells, are beneficial to cell uptake, have high fluorescence quantum yields in aqueous solutions after compounding, and can be applied in biological imaging. In addition, the carbon nanodot amino acid reagents can effectively reduce the number of cells and fat in the body, so that the effect of weight loss is achieved.

Description

Technical Field

[0001] This invention relates to the field of carbon nanomaterials technology, and more specifically, to a carbon nanodot composite amino acid reagent, its preparation method, and its application. Background Technology

[0002] Carbon dots (CDots) are an emerging class of luminescent carbon nanomaterials possessing excellent optical properties, low synthesis costs, no / low cytotoxicity, and good biocompatibility, giving them unique advantages as fluorescent markers and for drug delivery in bioimaging and other biomedical applications. However, reported red / near-infrared emitting CDots require complex fabrication processes and typically contain numerous negatively charged electron-withdrawing groups on their surface, making it difficult for them to bind to negatively charged cell membranes and penetrate cells, resulting in weak cellular uptake. This significantly hinders their application in bioimaging and biomedicine.

[0003] It is evident that existing methods for preparing carbon nanodots generally suffer from the following problem: the prepared carbon nanodots are not easily absorbed by organisms.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a carbon nanodot composite amino acid reagent, its preparation method, and its application, so as to improve the above-mentioned technical problems.

[0006] This invention is implemented as follows: In a first aspect, the present invention provides a carbon nanodot composite amino acid reagent, comprising carbon nanodots and amino acids composited together, wherein the carbon nanodots have electron-withdrawing groups on their surface.

[0007] Secondly, the present invention provides a method for preparing the above-mentioned carbon nanodot composite amino acid reagent, which includes: heating and combining the carbon nanodots having electron-withdrawing groups on their surface and the amino acid in a solution system.

[0008] Thirdly, the present invention also provides the application of the above-mentioned carbon nanodot composite amino acid reagent in the preparation of bioimaging reagents.

[0009] Fourthly, the present invention also provides the application of the above-mentioned carbon nanodot composite amino acid reagent in the preparation of weight-loss drugs.

[0010] This invention offers the following advantages: By combining carbon nanodots with electron-withdrawing groups on their surface with amino acids, the two are bonded together via hydrogen bonds to form a carbon nanodot amino acid reagent. This eliminates the need for complex purification procedures. The carbon nanodot amino acid reagent possesses the properties of both amino acids and carbon dots, readily binding to cell membranes and penetrating cells, facilitating cellular uptake. Furthermore, the composite exhibits a high fluorescence quantum yield in aqueous solution, making it applicable in bioimaging. In addition, this carbon nanodot amino acid reagent can effectively reduce the amount of fat in cells and the body, thereby achieving a weight-loss effect. Attached Figure Description

[0011] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0012] Figure 1 These are transmission electron microscopy (TEM) images of the carbon nanodot composite amino acid reagents (L-Arg@CDots, D-Arg@CDots, L-Lys@CDots) of Examples 1, 13, and 14 of this invention. Figure 2 The absorption spectra of the carbon nanodot composite amino acid reagents (L-Arg@CDots, D-Arg@CDots, L-Lys@CDots) in aqueous solution of Examples 1, 13 and 14 of this invention are shown. Figure 3 The emission spectra of the carbon nanodot composite amino acid reagents (L-Arg@CDots, D-Arg@CDots, L-Lys@CDots) of Examples 1, 13 and 14 of the present invention (under 550 nm laser excitation). Figure 4 Emission spectra of carbon nanodot composite amino acid reagents prepared at different concentrations of L-Arg in Examples 1, 13 and 14 of the present invention (under 550 nm laser excitation). Figure 5 This is a schematic diagram illustrating the synthesis of carbon nanodot composite amino acid reagents (L-Arg@CDots, D-Arg@CDots, L-Lys@CDots) in Examples 1, 13, and 14 of this invention. Figure 6 This is an experimental image showing the efficient intracellular imaging of the carbon nanodot composite amino acid reagent (L-Arg@CDots) in Example 1. Figure 7The figure shows the experimental phenomenon that the carbon nanodot composite amino acid reagent (L-Arg@CDots) in Example 1 can reduce intracellular lipids. Figure 8 The figure shows the experimental phenomenon in which the carbon nanodot composite amino acid reagent (L-Arg@CDots) of Example 1 reduced the body weight of mice. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0014] The carbon nanodot composite amino acid reagent proposed in this invention, its preparation method, and its application are described in detail below.

[0015] Some embodiments of the present invention provide a carbon nanodot composite amino acid reagent, which comprises carbon nanodots and amino acids that are composited together, wherein the carbon nanodots have electron-withdrawing groups on their surface.

[0016] By combining carbon nanodots with electron-withdrawing groups on their surface with amino acids, the electron-withdrawing groups on the carbon nanodots can act as hydrogen bond acceptors. The amino acids then bind to the carbon nanodots via hydrogen bonds. Without complex purification procedures, the resulting carbon nanodot-amino acid reagent possesses the properties of both amino acids and carbon dots, readily binding to cell membranes and penetrating cells for cellular uptake. Furthermore, the composite exhibits high fluorescence quantum yield in aqueous solution, making it suitable for applications in bioimaging. This carbon nanodot-amino acid composite reagent is highly water-soluble, biocompatible, has low cytotoxicity, and is excreted through the kidneys.

[0017] Numerous studies have shown that obesity increases the risk of various diseases, such as type 2 diabetes, high blood pressure, fatty liver disease, cancer, stroke, and myocardial infarction, thereby significantly reducing quality of life and life expectancy. Exercise is a common method for weight loss. A growing body of research indicates that diet and supplements can effectively control weight, thereby reducing the risk of obesity-related diseases.

[0018] The carbon nanodot amino acid reagents mentioned above can effectively reduce the number of cells and fat in the body, thereby achieving the effect of weight loss.

[0019] Specifically, in some embodiments, the mass ratio of carbon nanodots to amino acids is 1:(1~300), for example, the mass ratio can be 1:1, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190, 1:200, 1:210, 1:220, 1:230, 1:240, 1:250, 1:260, 1:270, 1:280, 1:290, or 1:300, preferably 1:(50~200). Controlling the mass ratio of carbon nanodots to amino acids within the above range is preferable, as it can further improve the fluorescence quantum yield of the composite material.

[0020] In some embodiments, the amino acid is selected from at least one of arginine, lysine, and histidine, all of which can form composite materials with carbon nanodots.

[0021] In a preferred embodiment, the amino acid is arginine, and more preferably, L-arginine. L-arginine (L-Arg) is an essential amino acid for the human body. Clinical studies have shown that L-Arg is an effective nutritional supplement that can reduce the amount of white fat in the body by increasing the oxidation of glucose and fatty acids. Intracellular uptake of L-Arg is achieved through the amino acid transport system γ. + L-Arg uptake is limited by the levels of cell membrane transport proteins CAT-2B and CAT-1. Therefore, combining L-Arg with carbon nanodots to enhance intracellular L-Arg uptake is of significant value.

[0022] In some embodiments, the carbon nanodot composite amino acid reagent is formed as nanoparticles with a particle size of 2-6 nm. Nanoparticles of this size have better uptake performance, easily enter cells, and have better fluorescence imaging effects.

[0023] Furthermore, in some embodiments, carbon nanodots are not limited to any particular source, as long as they have electron-withdrawing groups on their surface that can form hydrogen bonds with amino acids. For example, carbon nanodots include, but are not limited to, carbon dots prepared from citric acid and urea.

[0024] Furthermore, in some embodiments, the main absorption band of the carbon nanodot composite amino acid reagent in water is 520 nm to 600 nm, preferably 545 nm to 555 nm, for example, 550 nm. And / or, when excited by a light source in the ultraviolet-visible region, the emission peak of the carbon nanodot composite amino acid reagent is in the range of 500 nm to 800 nm, preferably 600 nm when excited at 550 nm.

[0025] Continuing on the above, some embodiments of the present invention also provide a method for preparing the carbon nanodot composite amino acid reagent described in the above embodiments, which includes: heating and combining carbon nanodots with electron-withdrawing groups on their surface with amino acids in a solution system. This method for preparing the carbon nanodot composite amino acid reagent is simple, inexpensive, and easy to mass-produce.

[0026] Specifically, to ensure a thorough and uniform reaction between carbon nanodots and amino acids, in some embodiments, the heating and compounding process is carried out in an aqueous solution. Carbon nanodots and amino acids are added to water in a specific ratio to form a mixed aqueous solution, which is then subjected to heating treatment. The concentration of amino acids in the aqueous solution is 5 mg / mL to 50 mg / mL.

[0027] In some embodiments, the heating and bonding temperature is 30℃~95℃, and the heating and bonding time is 5min~30min. It is understood that the heating and bonding temperature can be 30℃, 45℃, 45℃, 50℃, 60℃, etc., or any value between these adjacent temperature values, and the heating and bonding time can be 5min, 10min, 15min, 20min, 25min, 30min, etc., or any value between these adjacent time values.

[0028] At the above temperatures and heating times, amino acids and carbon nanodots can fully react and combine to form composite nanoparticles with uniform particle size and good fluorescence quantum yield.

[0029] It should be noted that in some embodiments, the above-mentioned carbon nanodots may be red-light emitting carbon nanodots. For example, these carbon nanodots are mainly prepared by a solvothermal reaction of citric acid and urea. Specifically, citric acid and urea are dissolved in dimethylformamide (DMF), then placed in a reaction vessel and heated at high temperature to carry out a solvothermal reaction, yielding the carbon nanodots. The mass ratio of citric acid to urea can be 1:2, and the temperature of the solvothermal reaction can be 150~200℃, for example, 160℃.

[0030] Furthermore, in some embodiments, the preparation method of carbon nanodot composite amino acid reagent further includes solid-liquid separation after heating and composite, and drying of the solid.

[0031] Specifically, the solid-liquid separation method is dialysis separation, and the drying method is freeze drying.

[0032] Some embodiments of the present invention also provide the application of the above-mentioned carbon nanoparticle composite amino acid reagent in the preparation of bioimaging reagents. Specifically, the above-mentioned carbon nanoparticle composite amino acid reagent can be applied in bioimaging, particularly in the fluorescence imaging of cells.

[0033] Some embodiments of the present invention also provide the application of carbon nanodot composite amino acid reagents in the preparation of weight-loss drugs. These carbon nanodot composite amino acid reagents can reduce fat in cells and animals.

[0034] It should be noted that the aqueous solution of this carbon nanodot composite amino acid reagent can be injected into animals via intraperitoneal or intravenous injection to reduce body fat.

[0035] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0036] Example 1 This embodiment provides a carbon nanodot composite amino acid reagent, the preparation process of which includes the following steps: 2g of citric acid and 4g of urea were dissolved in 20mL of dimethylformamide (DMF). The liquid was placed in a 50mL polytetrafluoroethylene high-pressure reactor and heated to 160℃ for 4 hours using an oven. After the reaction, 60mL of ethanol was added to the solution, and the mixture was centrifuged at 10000 rpm. The supernatant was removed, leaving a precipitate. This precipitate was centrifuged three times, and the lower precipitate was freeze-dried to obtain a black solid powder, namely carbon nanodots (CDs). The prepared CDs were mixed with water to obtain a CDs aqueous solution with a concentration of 1mg / mL. 1mL of the CDs aqueous solution was added to 1mL of L-Arg (L-arginine) aqueous solution with a concentration of 100mg / mL. The mixed solution was heated at 40℃ for 15 minutes. The resulting solution was placed in a dialysis bag (cutoff molecular weight 1000Da), dialyzed with ultrapure water for half a day, and then freeze-dried to obtain a carbon nanodot composite amino acid reagent.

[0037] Example 2 This embodiment provides a carbon nanodot composite amino acid reagent, which differs from Example 1 only in the preparation process: the concentration of the L-Arg aqueous solution is 10 mg / mL.

[0038] Example 3 This embodiment provides a carbon nanodot composite amino acid reagent, which differs from Example 1 only in the preparation process: the concentration of the L-Arg aqueous solution is 20 mg / mL.

[0039] Example 4 This embodiment provides a carbon nanodot composite amino acid reagent, which differs from Example 1 only in the preparation process: the concentration of the L-Arg aqueous solution is 30 mg / mL.

[0040] Example 5 This embodiment provides a carbon nanodot composite amino acid reagent, which differs from Example 1 only in the preparation process: the concentration of the L-Arg aqueous solution is 40 mg / mL.

[0041] Example 6 This embodiment provides a carbon nanodot composite amino acid reagent, which differs from Example 1 only in the preparation process: the concentration of the L-Arg aqueous solution is 50 mg / mL.

[0042] Example 7 This embodiment provides a carbon nanodot composite amino acid reagent, which differs from Example 1 only in the preparation process: the concentration of the L-Arg aqueous solution is 60 mg / mL.

[0043] Example 8 This embodiment provides a carbon nanodot composite amino acid reagent, which differs from Example 1 only in the preparation process: the mixed solution is heated at 40°C for 5 minutes.

[0044] Example 9 This embodiment provides a carbon nanodot composite amino acid reagent, which differs from Example 1 only in the preparation process: the mixed solution is heated at 45°C for 10 minutes.

[0045] Example 10 This embodiment provides a carbon nanodot composite amino acid reagent, which differs from Example 1 only in the preparation process: the mixed solution is heated at 50°C for 5 minutes.

[0046] Example 11 This embodiment provides a carbon nanodot composite amino acid reagent, which differs from Example 1 only in the preparation process: the mixed solution is heated at 60°C for 10 minutes.

[0047] Example 12 This embodiment provides a carbon nanodot composite amino acid reagent, which differs from Example 1 only in the preparation process: the concentration of the carbon nanodot aqueous solution is 2 mg / mL, and the concentration of the L-Arg aqueous solution is 100 mg / mL.

[0048] Example 13 The only difference from Example 1 is in the preparation process: L-arginine is replaced with D-arginine.

[0049] Example 14 The only difference from Example 1 is in the preparation process: L-arginine is replaced with L-lysine.

[0050] Experimental Example 1 Transmission electron microscopy (TEM) analysis was performed on the carbon nanodot composite amino acid reagents (L-Arg@CDots, D-Arg@CDots, L-Lys@CDots) obtained in Examples 1, 13, and 14. The TEM images are shown below. Figure 1 As shown.

[0051] Depend on Figure 1 It is known that the particle size of the carbon nanodot composite amino acid reagent is about 2-6 nm, with an average size of about 5 nm.

[0052] Experimental Example 2 The optical properties of the carbon nanodot composite amino acid reagents (L-Arg@CDots, D-Arg@CDots, L-Lys@CDots) obtained in Examples 1, 13, and 14 were observed by absorption and fluorescence spectroscopy (PL). Figure 2 and Figure 3 As shown.

[0053] Depend on Figure 2 It can be seen that in aqueous solution, carbon nanodots exhibit a major absorption band at 556 nm, while the carbon nanodot-amino acid reagent shows a certain blue shift, from 556 nm to 550 nm, and the intensity of the absorption peak is greatly improved. Under 550 nm excitation, the emission peak of L-Arg@CDots is at 600 nm. The red emission of CDs in water is relatively weak, with a PLQY of 2%, while the intensity of L-Arg@CDots can be increased by 12 times, with a PLQY of 24%.

[0054] Depend on Figure 4 It can be seen that different concentrations of L-Arg aqueous solution have a certain impact on the performance of carbon nanodot composite amino acid reagent. As the concentration of L-Arg aqueous solution increases, the fluorescence intensity of carbon nanodot composite amino acid reagent also gradually increases. When the concentration of carbon nanodots and L-Arg aqueous solution reaches 1:130, the fluorescence intensity reaches the highest level, at which point the binding strength between L-Arg aqueous solution and carbon nanodots is the strongest.

[0055] Experimental Example 3 By analyzing the binding mechanisms of different amino acids and carbon dots to form complexes, such as... Figure 4 As shown.

[0056] Depend on Figure 5It is known that L-Arg exhibits stronger bonding strength on CDots surfaces than D-Arg and L-Lys. This is because the surface of CDots is rich in electron-withdrawing groups, such as -COOH and C=O, which generate red emission centers on the carbon dots. The bonding strength of these amino acids on the CDots surface is determined by the number and angle of hydrogen bonds on its surface. The main difference between L-Lys and D / L-Arg is that L-Lys contains an NH2 group at one end, while D / L-Arg contains a guanidinium group. The NH2 group can form a single hydrogen bond, while the guanidinium group can form stronger multiple hydrogen bonds, resulting in stronger bonding strength of D / L-Arg@CDots than L-Lys@CDots. Based on molecular simulations, the steric structure of L-Arg is more curved than that of D-Arg. Considering the directionality of hydrogen bonds, the more curved L-Arg is more conducive to forming stronger hydrogen bonds at both ends on the CDots surface compared to D-Arg.

[0057] Test Example 4 The staining performance of L-Arg@CDots cells obtained in Example 1 was tested.

[0058] Figure 6 Hepa1-6 cells incubated with L-Arg@CDots for 1.5 hours exhibited clear red fluorescence under 543nm laser excitation. High-magnification magnification revealed that L-Arg@CDots were primarily distributed in the cytoplasm. Conversely, very weak red fluorescence signals were observed in cells incubated with CDots for 1.5 hours. Therefore, the nanodot complex provided in this embodiment of the invention can be applied to bioimaging.

[0059] Experimental Example 5 The performance of L-Arg@CDots obtained in Example 1 in reducing intracellular lipids was tested.

[0060] Figure 7 The diagram illustrates the investigation of oleic acid (OA)-induced adipogenesis in Hepa1-6 cells. Cells were treated with 0.1 mM OA for 12 h, followed by treatment with CDots, L-Arg, D-Arg, L-Arg@CDots (Example 1), and D-Arg@CDots (Example 13) for 12 h, with CDots concentrations of 20 μg / mL. -1 The L-Arg concentration was 2 mg / mL. -1The cells were then labeled with the fluorescent dye BODIPY 493 / 503 to represent lipids. Green fluorescence was observed after incubation with OA, indicating significant lipid accumulation in the OA-treated cells. No significant change in green fluorescence was observed in the CDots-treated group of OA-treated cells, indicating that CDots had no effect on intracellular lipids. Treatment with L-Arg weakened the green fluorescence signal of lipids in OA-treated cells. Conversely, treatment with L-Arg@CDots significantly weakened the green fluorescence signal of lipids in OA-treated cells, indicating much less lipid accumulation. Therefore, the nanodot complex provided in this embodiment can be used to reduce cellular lipids.

[0061] Experimental Example 6 The performance of L-Arg@CDots obtained in Example 1 in reducing body fat in animals was tested.

[0062] Figure 8 It is known that intraperitoneal injection of L-Arg@CDots can inhibit fat production in mice, thereby causing them to lose weight. Therefore, the nanodot composite provided in this embodiment of the invention can be used for weight loss.

[0063] In summary, by combining carbon nanodots with hydrogen bond acceptor groups on their surface with amino acids, a carbon nanodot-amino acid composite formulation with enhanced fluorescence quantum yield can be prepared without complex purification procedures. Among these, arginine-modified carbon nanodots exhibit the highest increase in fluorescence quantum yield in aqueous solution and can also enhance intracellular uptake of arginine, thereby reducing intracellular lipid production. Injection of mice with the carbon nanodot-arginine composite formulation reduced weight gain by inhibiting food intake and shrinking white adipose tissue cells, thus significantly inhibiting obesity. This demonstrates that the carbon nanodot-amino acid composite formulation of this invention can effectively reduce the amount of fat in cells and within animals, achieving a weight loss effect.

[0064] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A carbon nanodot composite amino acid reagent, characterized in that, It comprises carbon nanodots and amino acids that are composite together, wherein the carbon nanodots have electron-withdrawing groups on their surface; The carbon nanodots and the amino acid are connected by hydrogen bonds; The mass ratio of the carbon nanodots to the amino acid is 1:(50-200), and the amino acid is arginine. The carbon nanodot composite amino acid reagent is a nanoparticle with a particle size of 2-6 nm. The carbon nanodots are carbon dots prepared from citric acid and urea. The main absorption band of the carbon nanodot composite amino acid reagent in water is 545-555 nm. The carbon nanodot composite amino acid reagent exhibits an emission peak in the ultraviolet-visible light region when excited by a light source. The emission peak is between 500 nm and 800 nm.

2. The carbon nanodot composite amino acid reagent according to claim 1, characterized in that, The amino acid in question is L-arginine.

3. The carbon nanodot composite amino acid reagent according to claim 1, characterized in that, The carbon nanodot composite amino acid reagent exhibits an emission peak at 600 nm when excited by a 550 nm light source in the ultraviolet-visible region.

4. A method for preparing a carbon nanodot composite amino acid reagent as described in any one of claims 1 to 3, characterized in that, It includes: The carbon nanodots with electron-withdrawing groups on their surface and the amino acid are thermally combined in a solution system.

5. The preparation method according to claim 4, characterized in that, The heating and compounding process is carried out in an aqueous solution. The concentration of amino acids in the aqueous solution is 5 mg / mL. -1 ~50mg mL -1 ; The heating temperature for composite bonding is 30℃~95℃, and the heating time is 5min~30min.

6. The preparation method according to claim 4 or 5, characterized in that, It also includes solid-liquid separation after heating and compounding, and drying of the solid; The solid-liquid separation method is dialysis separation, and the drying method is freeze drying.

7. The application of the carbon nanodot composite amino acid reagent as described in any one of claims 1 to 3 in the preparation of cell fluorescence imaging reagents.

8. The application of the carbon nanodot composite amino acid reagent as described in any one of claims 1 to 3 in the preparation of weight-loss drugs, characterized in that, The amino acid selected for the carbon nanodot composite amino acid reagent is arginine.

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