A luteolin carbon dot guided to lysosome and a preparation method and application thereof

Abstract

The application discloses the technical field of carbon dot application, and relates to a luteolin carbon dot for guiding lysosomes and a preparation method and application thereof. The luteolin carbon dot is prepared by using luteolin and 2,5-dihydroxyterephthalic acid as carbon source precursors through a hydrothermal method. The prepared luteolin carbon dot has a particle size of 0.5-4 nm, a fluorescence excitation wavelength of 360-440 nm, a maximum emission wavelength of 540 nm and a Zeta potential of -19 mv. Research shows that the luteolin carbon dot can guide macrophage and adipocyte lysosomes, promote M2 type macrophage polarization, and up-regulate the expression of the heat production and brown colorization marker gene Ucp1 of beige adipocytes. Further, the M2 type macrophage loaded with the luteolin carbon dot can promote the expression of the Ucp1 of the beige adipocyte through the release of the carbon dot and the paracrine effect, thereby increasing heat production and energy consumption. The application lays a foundation for developing an intervention and treatment scheme for obesity and related metabolic diseases based on the carbon dot in the future.

Description

Technical Field

[0001] This invention relates to the field of carbon dot application technology, specifically to a luteolin carbon dot for guiding lysosomes, its preparation method, and its application. Background Technology

[0002] Obesity is a prevalent metabolic disease worldwide, its core being the excessive accumulation of body fat. In white adipose tissue, white adipocytes are responsible for energy storage, and their abnormal accumulation is a major contributing factor to obesity. Beige adipocytes are also distributed within white adipose tissue and highly express thermogenesis and browning marker genes. Ucp1 M2 macrophages can convert chemical energy into heat energy, which is then consumed. Activation can further increase browning and heat production. Furthermore, in adipose tissue, M2 macrophages can also promote browning of beige adipocytes by parasecrinating various substances such as catecholamines and Slit3 protein. Therefore, inducing M2 macrophage polarization and driving adipocyte browning through M2 macrophages could be developed into an interventional treatment for obesity and related metabolic diseases.

[0003] Luteolin is a common dietary flavonoid compound that can regulate lysosomal activity, induce a moderate increase in intracellular ROS and lysosomal-mitochondrial interactions, thereby promoting macrophage M2 polarization. Furthermore, luteolin can also regulate lysosomes to maintain mitochondrial homeostasis in adipocytes, activate the AMPK / PGC1α signaling pathway, and upregulate thermogenesis and browning marker genes in beige adipocytes. Ucp1 However, luteolin itself has poor water solubility, making it difficult to fully dissolve in aqueous solutions. This results in ineffective dispersion and dissolution in the gastrointestinal tract, significantly impacting oral absorption efficiency. Furthermore, luteolin exhibits strong biotoxicity; high doses can easily irritate and damage organs such as the gastrointestinal tract and liver, leading to increased cytotoxicity. Luteolin also has weak cell membrane penetration, hindering its efficient entry into target cells such as adipocytes and macrophages, greatly limiting its practical application in improving obesity.

[0004] Carbon dots (CDs) are a new type of carbon-based nanomaterials with a size of less than 10 nm. They have the advantages of good water solubility, biocompatibility, high stability and easy functionalization. Carbon dots have shown broad application prospects in the fields of biomedicine, such as targeted delivery and drug carriers, and can effectively enhance the bioavailability and targeting of natural active substances.

[0005] In summary, if luteolin is formulated into carbon dots and directed to lysosomes, it can not only overcome its poor water solubility and low bioavailability, but also achieve adipocyte browning driven by M2 macrophages; thereby increasing thermogenesis and energy expenditure, laying the foundation for developing interventional treatments for obesity and related metabolic diseases. A search of existing literature and published patents has not yet revealed any lysosome-directed luteolin carbon dots or their applications. Summary of the Invention

[0006] The purpose of this invention is to solve the above-mentioned problems by providing a luteolin carbon dot for guiding lysosomes, its preparation method and application, especially its application in the browning of beige adipocytes driven by M2 macrophages.

[0007] The present invention achieves the above objectives through the following technical solutions:

[0008] As a first aspect of the present invention, a luteolin carbon dot is provided for guiding lysosomes, the luteolin carbon dot being prepared by hydrothermal reaction using luteolin and 2,5-dihydroxyterephthalic acid as carbon source precursors; the luteolin carbon dot specifically guides to the lysosomes of macrophages and adipocytes.

[0009] As a further optimization of the present invention, the luteolin carbon dots are spherical particles with a particle size of 0.5-4 nm; the fluorescence excitation wavelength of the luteolin carbon dots is 360-440 nm, the maximum emission wavelength is 540 nm, and the zeta potential is -19 mV.

[0010] As a further optimization of the present invention, the preparation method is as follows: luteolin and 2,5-dihydroxyterephthalic acid are dispersed in deionized water at a molar ratio to obtain a mixed solution. The mixed solution is subjected to a hydrothermal reaction. After the hydrothermal reaction, the solution is filtered and dialyzed to obtain a carbon dot solution. The carbon dot solution is freeze-dried to obtain luteolin carbon dots that guide lysosomes.

[0011] As a further optimization of the present invention, the molar ratio of luteolin and 2,5-dihydroxyterephthalic acid is 1:1-3; the hydrothermal reaction temperature is 160-260 ℃, and the reaction time is 2-10 h; the pore size of the filter membrane in the filtration treatment is 0.22 μm; and the size of the dialysis bag in the dialysis treatment is 1000 Da.

[0012] As a second aspect of the present invention, the application of luteolin carbon dots as described in any one of the above claims in lysosome-targeted fluorescence imaging is also provided, wherein the luteolin carbon dots serve as green fluorescent probes to guide lysosomes enriched in macrophages and adipocytes for fluorescence imaging of lysosomes in macrophages and adipocytes.

[0013] As a third aspect of the present invention, the application of luteolin carbon dots as described in any of the above claims in the preparation of agents that promote M2 macrophage polarization is also provided, wherein macrophages are treated with luteolin carbon dots to upregulate M2 macrophage marker genes. Arg1 The mRNA expression level of macrophages was increased, thereby increasing the proportion of the M2 subset of macrophages and promoting macrophage polarization towards the M2 type.

[0014] As a third aspect of the present invention, the application of luteolin carbon dots as described in any of the above claims in the preparation of an agent that promotes browning of beige adipocytes is also provided. By treating beige adipocytes with luteolin carbon dots, the thermogenic and browning marker genes of beige adipocytes are upregulated. Ucp1 The mRNA expression level was adjusted to promote browning of beige adipocytes.

[0015] As a fourth aspect of the invention, the application of luteolin-loaded M2 macrophages in the preparation of an agent promoting browning of beige adipocytes is also provided. The macrophages are pretreated with luteolin carbon dots as described above to induce M2 polarization. The pretreated M2 macrophages are then co-cultured with beige adipocytes. The release of the loaded luteolin carbon dots and paracrine effects indirectly upregulate beige adipocytes. Ucp1 Gene expression promotes its browning.

[0016] As a further optimization of the present invention, the concentration of luteolin carbon dots used to pretreat macrophages is 200 μg / mL, the treatment time is 24 h, and the co-culture time is 24 h.

[0017] The beneficial effects of this invention are as follows:

[0018] (1) The preparation process of luteolin carbon dots in this invention is simple. It is prepared by hydrothermal method. The prepared luteolin carbon dots have good water solubility and low toxicity, which solves the problem of poor water solubility and low bioavailability of luteolin.

[0019] (2) The luteolin carbon dots prepared in this invention are spherical particles with an average particle size of 0.5-4 nm; the fluorescence excitation wavelength is 360-440 nm and the maximum emission wavelength is 540 nm; the zeta potential is -19 mV; this particle size is conducive to the carbon dots entering the cell, its fluorescence characteristics are conducive to tracking the distribution of carbon dots in the cell, and the negative charge of the zeta potential is conducive to guiding them to the lysosomes of the cell.

[0020] (3) The luteolin carbon dots in this invention can promote M2 macrophage polarization and upregulate the thermogenic genes of beige adipocytes. Ucp1 It expresses and promotes browning of adipocytes, and can be loaded into M2 macrophages, promoting the browning of beige adipocytes through carbon dot release and paracrine effects. Ucp1 The expression of this carbon point increases heat production and energy consumption, laying the foundation for future intervention and treatment programs for obesity and related metabolic diseases based on this carbon point. Attached Figure Description

[0021] Figure 1 A schematic diagram illustrating the construction and application of luteolin carbon dots provided by this invention;

[0022] Figure 2 Transmission electron microscopy image (a) and particle size distribution (b) of luteolin carbon dots prepared in Example 1 are provided for the present invention.

[0023] Figure 3 The Fourier transform infrared spectrum of the carbon dots of luteolin prepared in Example 1 is provided by the present invention;

[0024] Figure 4 The fluorescence spectrum of luteolin carbon dots obtained in Example 1 is provided by the present invention;

[0025] Figure 5 The effect of different concentrations of luteolin prepared in Example 1 on the cell survival of RAW 264.7 cells after 24 h of carbon dot treatment;

[0026] Figure 6 This is a graph showing the effect of different concentrations of luteolin prepared in Example 1 on the cell survival of C3H10T1 / 2 cells after carbon dot treatment for 24 h.

[0027] Figure 7 This is a lysosomal guided colocalization fluorescence imaging image of luteolin carbon dots obtained in Example 1 in RAW 264.7 cells and C3H10T1 / 2 cells;

[0028] Figure 8 Macrophages were treated with different concentrations of luteolin carbon dots prepared in Example 1 for 24 h. Arg1 Relative mRNA expression map;

[0029] Figure 9 This is a flow cytometry quantification image of macrophages after treatment with carbon dots of luteolin prepared in Example 1 at a concentration of 200 μg / mL for 24 h.

[0030] Figure 10 After treating beige adipocytes with different concentrations of luteolin carbon dots prepared in Example 1 for 24 hours... Ucp1 Relative mRNA expression map;

[0031] Figure 11The macrophages treated with 200 μg / mL luteolin carbon dots as prepared in Example 1 for 24 h were co-cultured with beige adipocytes for 24 h. Ucp1 Relative mRNA expression map. Detailed Implementation

[0032] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0033] Unless otherwise specified, all experimental methods used in the following examples can be performed using conventional methods. All materials used, unless otherwise specified, are commercially available.

[0034] Example 1

[0035] This embodiment provides a method for preparing luteolin carbon dots using a hydrothermal method. The specific operation steps are as follows:

[0036] (1) Accurately weigh 0.25 g of luteolin and 0.173 g of 2,5-dihydroxyterephthalic acid (molar ratio of the two is 1:1) and place them in a 50 mL centrifuge tube. Then add 20 mL of deionized water, sonicate for 20 min to obtain a mixed solution.

[0037] (2) Transfer the mixed solution obtained in step (1) to a 100 mL polytetrafluoroethylene high-pressure reactor, and then place it in an electric thermostatic drying oven for hydrothermal reaction. Set the temperature to 180 °C and the reaction time to 6 h. After the reaction is completed, let it cool naturally to room temperature, then take out the carbon dot solution for subsequent purification.

[0038] (3) Transfer the carbon dot solution obtained in step (2) to a 50 mL centrifuge tube, centrifuge at 12000 g for 15 min using a high-speed centrifuge, and take the supernatant for further filtration with a 0.22 μm filter;

[0039] (4) Transfer the filtered carbon dot solution to a 1000 Da dialysis bag and dialyze for 72 h for purification. Use deionized water for dialysis and change the water every 12 h.

[0040] (5) Freeze the carbon dot solution purified in step (4) and freeze it for 48 hours to obtain carbon dot solid, which is luteolin carbon dot.

[0041] Example 2

[0042] This embodiment provides a method for preparing luteolin carbon dots using a hydrothermal method. The specific operation steps are as follows:

[0043] (1) Accurately weigh 0.25 g of luteolin and 0.519 g of 2,5-dihydroxyterephthalic acid (molar ratio of the two is 1:3) and place them in a 50 mL centrifuge tube. Then add 20 mL of deionized water, sonicate for 20 min to obtain a mixed solution.

[0044] (2) Transfer the mixed solution obtained in step (1) to a 100 mL polytetrafluoroethylene high-pressure reactor, and then place it in an electric thermostatic drying oven for hydrothermal reaction. Set the temperature to 260 °C and the reaction time to 2 h. After the reaction is completed, let it cool naturally to room temperature, then take out the carbon dot solution for subsequent purification.

[0045] (3) Transfer the carbon dot solution obtained in step (2) to a 50 mL centrifuge tube, centrifuge at 12000 g for 15 min using a high-speed centrifuge, and take the supernatant for further filtration with a 0.22 μm filter;

[0046] (4) Transfer the filtered carbon dot solution to a 1000 Da dialysis bag and dialyze for 72 h for purification. Use deionized water for dialysis and change the water every 12 h.

[0047] (5) Freeze the carbon dot solution purified in step (4) and freeze it for 48 h using a freeze dryer to obtain carbon dot solid, which is luteolin carbon dot.

[0048] Example 3

[0049] This embodiment characterizes the luteolin carbon dots prepared in Example 1.

[0050] Luteolin carbon dots were evenly dispersed in water, exhibiting good water dispersibility; from Figure 2 The transmission electron microscopy image of a shows that the carbon dots of luteolin are well-dispersed spherical particles with no obvious aggregation. Figure 2 b shows that the particle size distribution of the carbon dots is 0.5-4 nm; Figure 3 Fourier transform infrared spectra show that the carbon dots of luteolin are modified with functional groups such as hydroxyl and carbonyl groups. Figure 4The fluorescence spectrum of the luteolin carbon dot solution is shown in the figure. Typical carbon dot fluorescence spectra can be observed. Within the excitation wavelength range of 360-440 nm, the fluorescence emission peak intensity of the luteolin carbon dots first increases and then decreases with increasing excitation wavelength, reaching a maximum emission wavelength of 540 nm at an excitation wavelength of 380 nm, corresponding to green fluorescence. The zeta potential of the luteolin carbon dots is -19 mV, a negative potential that facilitates the orientation of the carbon dots to positively charged lysosomes.

[0051] Example 4

[0052] This embodiment conducts a cytotoxicity experiment on the luteolin carbon dots prepared in Example 1.

[0053] According to the standard protocol, the toxicity of luteolin carbon dot solution to RAW 264.7 cells (Cell Culture Center, Peking Union Medical College) and C3H10T1 / 2 cells (Cell Culture Center, Peking Union Medical College) was detected by the MTT assay. The specific steps are as follows:

[0054] First, the cells were arranged at a ratio of 1×10 4 Cells were seeded per well in sterile 96-well plates and cultured for 24 h. Then, different concentrations of luteolin carbon dot solution (final concentrations of 0, 50, 100, and 200 μg / mL, respectively) dispersed in PBS were added to the 96-well plates, and incubation continued for another 24 h. On the second day, the mixed culture medium was aspirated, and cytotoxicity was determined using the MTT assay. After 4 h, 150 μL DDMSO was added to each well to dissolve the purple crystals at the bottom of the plate. The plates were shaken at 150 rpm for 10 minutes to completely dissolve the crystals. Finally, the absorbance of the purple solution in the 96-well plate was measured at 490 nm using a microplate reader.

[0055] Figure 5 and Figure 6 Cell viability was shown after 24 h of treatment with different concentrations of luteolin carbon dots. The cytotoxicity test results showed that different concentrations of luteolin carbon dots did not harm cells and could even promote cell proliferation to some extent. These data indicate that luteolin carbon dots exhibit low cytotoxicity.

[0056] Example 5

[0057] This embodiment demonstrates a guided lysosomal imaging experiment on the luteolin carbon dots prepared in Example 1.

[0058] RAW 264.7 cells and C3H10T1 / 2 cells were mixed at a ratio of 1×10⁻⁶. 5Each sample was inoculated into a laser confocal microscopy dish, and then luteolin carbon dots (200 μg / mL) were added and co-cultured for 12 h. After washing three times with PBS, a commercial fluorescent lysosomal fluorescent probe was added and co-incubated for 30 min. Fluorescence images were taken by laser confocal microscopy under 405 nm excitation, with a scale bar of 20 μm.

[0059] The results are as follows Figure 7 The results showed that the luteolin carbon dots (green fluorescent signal) and the commercial lysosomal fluorescent probe (red fluorescent signal) had a clear overlapping distribution in the cell. The green fluorescence of the luteolin carbon dots was precisely concentrated in the lysosomal region marked by the red probe, indicating that the carbon dots could successfully reach and accumulate in the lysosomes of the cell. This guiding ability provides support for its subsequent function in regulating macrophage M2 polarization and beige adipocyte browning.

[0060] Example 6

[0061] This embodiment uses the luteolin carbon dots obtained in Example 1 to promote M2 polarization of macrophages.

[0062] Five groups were set up in the experiment: the control group consisted of normally cultured RAW 264.7 cells; the positive control group was induced with 10 ng / mL interleukin-4; and the experimental groups were treated with luteolin carbon dots at concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively. All groups were treated for 24 h, and then marker genes of M2 macrophages were detected. Arg1 mRNA expression level of (NCBI Gene ID: 11846).

[0063] like Figure 8 As shown, compared with the control group, the mRNA expression level of Arg1, the M2 type marker gene, in macrophages was significantly increased after interleukin-4 treatment, indicating that interleukin-4 can effectively induce macrophages to polarize towards the M2 type, thus verifying the reliability of the experimental system.

[0064] After treatment with 50 μg / mL, 100 μg / mL, and 200 μg / mL luteolin carbon dots, the Arg1 expression level of macrophages was significantly upregulated compared with the control group, indicating that luteolin carbon dots can effectively promote macrophage polarization to M2 type, and the 200 μg / mL concentration group had the most significant effect.

[0065] Furthermore, macrophages were treated alone with 200 μg / mL and analyzed by flow cytometry; the results are as follows. Figure 9As shown, the flow cytometry quantitative diagram reveals the changes in the macrophage M2 subset: the proportion of the M2 subset was low in the control group, the proportion of this subset increased after interleukin 4 treatment, and the proportion of the M2 subset further increased after treatment with 200 μg / mL luteolin carbon dots; the above results indicate that luteolin carbon dots with lysosome-guided properties can promote the polarization of M2 macrophages.

[0066] Example 7

[0067] This embodiment uses luteolin carbon dots obtained in Example 1 to promote browning of beige adipocytes. Five groups were set up: an uninduced group (C3H10T1 / 2 cells not induced into beige adipocytes); an induced group (cells induced to differentiate into beige adipocytes); and experimental groups treated with luteolin carbon dots at concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively. All groups were treated for 24 h, and then the thermogenicity and browning marker genes of beige adipocytes were detected. Ucp1 mRNA expression level of (NCBI Gene ID: 22227).

[0068] The results are as follows Figure 10 As shown, compared with the non-induced group, the induced group had Ucp1 The mRNA expression level was significantly upregulated, indicating that beige adipocytes were successfully induced to differentiate; compared with the induction group, after treatment with 50 μg / mL and 100 μg / mL luteolin carbon dots, Ucp1 The mRNA expression level was not significantly increased; however, when the concentration was increased to 200 μg / mL, the expression level was significantly increased. Ucp1 The expression level was significantly upregulated; the above results indicate that 200 μg / mL luteolin carbon dots can effectively promote browning of beige adipocytes.

[0069] Example 8

[0070] In this embodiment, the luteolin carbon dots obtained in Example 1 were loaded onto M2 macrophages to promote M2 macrophage polarization.

[0071] RAW 264.7 cells were seeded in the upper layer of a Transwell 24-well plate and treated for 24 h; C3H10T1 / 2 cells were seeded in a standard 24-well plate and induced to differentiate into mature beige adipocytes for 8 days. RAW 264.7 cells and beige adipocytes were then co-cultured for 24 h; subsequently, the mRNA expression level of the browning marker gene Ucp1 in beige adipocytes was detected.

[0072] The experiment was divided into four groups: the uninduced group, which consisted of C3H10T1 / 2 cells that were not induced into beige adipocytes; the induced group, which consisted of normally cultured RAW 264.7 cells co-cultured with beige adipocytes; the interleukin 4+ induced group, which was a positive control group, in which RAW 264.7 cells treated with 10 ng / ml interleukin 4 were co-cultured with beige adipocytes; and the luteolin carbon dot+ induced group, as described in Examples 6 and 7, which consisted of RAW 264.7 cells treated with 200 μg / mL luteolin carbon dots co-cultured with beige adipocytes.

[0073] The results are as follows Figure 11 As shown, the induction group Ucp1 The expression was significantly upregulated compared to the uninduced group, indicating successful differentiation of beige adipocytes; while the interleukin 4+ induced group and the luteolin carbon dot+ induced group showed a further significant increase in Ucp1 expression compared to the induced group; indicating that M2 macrophages loaded with this carbon dot can indirectly promote the browning of beige adipocytes in a co-culture system with beige adipocytes by releasing the loaded luteolin carbon dots and through paracrine effects.

[0074] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A luteolin carbon dot for guiding lysosomes, characterized in that, The luteolin carbon dots are prepared by hydrothermal reaction using luteolin and 2,5-dihydroxyterephthalic acid as carbon source precursors, with a molar ratio of luteolin to 2,5-dihydroxyterephthalic acid of 1:1-3; the hydrothermal reaction temperature is 160-260 °C, and the reaction time is 2-6 h; the luteolin carbon dots are specifically directed to the lysosomes of macrophages and adipocytes.

2. The luteolin carbon dot for guiding lysosomes according to claim 1, characterized in that, The luteolin carbon dots are spherical particles with a particle size of 0.5-4 nm; the fluorescence excitation wavelength of the luteolin carbon dots is 360-440 nm, the maximum emission wavelength is 540 nm, and the zeta potential is -19 mV.

3. A method for preparing luteolin carbon dots for guided lysosomes as described in any one of claims 1-2, characterized in that, The preparation method is as follows: luteolin and 2,5-dihydroxyterephthalic acid are dispersed in deionized water at a molar ratio to obtain a mixed solution. The mixed solution is subjected to a hydrothermal reaction. After the hydrothermal reaction, the solution is filtered and dialyzed to obtain a carbon dot solution. The carbon dot solution is freeze-dried to obtain luteolin carbon dots that guide lysosomes.

4. The method for preparing luteolin carbon dots for guided lysosomes according to claim 3, characterized in that, The filter membrane used in the filtration process has a pore size of 0.22 μm; the dialysis bag used in the dialysis process has a size of 1000 Da.

5. The application of luteolin carbon dots as described in any one of claims 1-2 in lysosomal targeted fluorescence imaging of cells not for disease diagnosis and treatment, characterized in that, The luteolin carbon dots serve as green fluorescent probes, guiding lysosomes enriched in macrophages and adipocytes for fluorescence imaging of lysosomes in macrophages and adipocytes.

6. The application of luteolin carbon dots as described in any one of claims 1-2 in the preparation of agents that promote macrophage M2 polarization, characterized in that, Treatment of macrophages with luteolin carbon dots upregulates M2 macrophage marker genes. Arg1 The mRNA expression level of macrophages was increased, thereby increasing the proportion of the M2 subset of macrophages and promoting macrophage polarization towards the M2 type.

7. The application of luteolin carbon dots as described in any one of claims 1-2 in the preparation of an agent promoting browning of beige adipocytes, characterized in that, Treatment of beige adipocytes with luteolin carbon dots upregulated thermogenesis and browning marker genes in beige adipocytes. Ucp1 The mRNA expression level was adjusted to promote browning of beige adipocytes.

8. The application of M2 macrophages loaded with luteolin in the preparation of an agent promoting browning of beige adipocytes, characterized in that, Macrophages were pretreated with luteolin carbon dots as described in any one of claims 1-2 to induce M2 polarization. The pretreated M2 macrophages were then co-cultured with beige adipocytes. The release of the loaded luteolin carbon dots and paracrine effects indirectly upregulated beige adipocytes. Ucp1 Gene expression promotes its browning.

9. The application according to claim 8, characterized in that, The concentration of luteolin carbon dots used to pretreat macrophages was 200 μg / mL, the treatment time was 24 h, and the co-culture time was 24 h.

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