A gold nanoflower enhanced flavonoid compound, a preparation method and application thereof

Abstract

The application provides a gold nanoflower enhanced flavonoid compound and a preparation method and application thereof, and the preparation method comprises the following steps: (1) mixing chloroauric acid, ascorbic acid, a protective agent and water to obtain a gold nanoflower solution; (2) mixing the gold nanoflower solution obtained in the step (1) with a flavonoid compound solution to obtain a gold nanoflower-flavonoid compound complex solution; and (3) mixing the gold nanoflower-flavonoid compound complex solution obtained in the step (2) with a biotin-PEG-SH solution to obtain the gold nanoflower enhanced flavonoid compound. The gold nanoflower enhanced flavonoid compound provided by the application effectively enhances the fluorescence intensity of a flavonoid traditional Chinese medicine small molecule at a tumor site, provides a more accurate and clear tumor image, and has the advantages of good targeting effect.

Description

[0001] This application claims priority to a Chinese patent application filed on April 11, 2024, with application number 202410436126.7, entitled “A Gold Nanoflower Enhanced Flavonoid Compound, Preparation Method and Application Thereof,” the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the technical field of nano-biomedical materials, and specifically relates to a gold nanoflower-enhanced flavonoid compound, a preparation method and application thereof, and especially relates to a gold nanoflower-enhanced flavonoid compound with good imaging effect, a preparation method and application thereof. Background Art

[0003] Malignant tumors (cancer) are among the most serious diseases that endanger human health, and the diagnosis and treatment of cancer are undoubtedly a major focus of modern medical research. Bioluminescence imaging has shown great potential in tumor diagnosis and treatment, with advantages including non-invasiveness, high sensitivity, and high resolution. By labeling tumors with fluorescent substances or markers, bioluminescence imaging can provide more accurate and clear images of tumors, facilitating applications such as early diagnosis, surgical navigation, and drug therapy. With the continued development and innovation of fluorescent probe technology, the prospects for bioluminescence imaging in tumor treatment will be even broader.

[0004] Active ingredients in traditional Chinese medicines (TCMs) are diverse in structure and complex in function. Many small molecules in TCMs can fluoresce under excitation light, but the fluorescence intensity is generally weak and the luminescence efficiency is low. Therefore, developing a solution that can enhance the fluorescence intensity of small molecules in TCMs while achieving excellent targeting effects has become an urgent challenge. Summary of the Invention

[0005] To address the shortcomings of the prior art, the present invention aims to provide a gold nanoflower-enhanced flavonoid compound, its preparation method, and its application, particularly a gold nanoflower-enhanced flavonoid compound with excellent imaging effects, its preparation method, and its application. The gold nanoflower-enhanced flavonoid compound provided by the present invention effectively enhances the fluorescence intensity of flavonoid-containing small molecules in traditional Chinese medicine at the tumor site, providing more accurate and clear tumor images and having the advantage of good targeting.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing gold nanoflower-enhanced flavonoid compounds, the preparation method comprising the following steps:

[0008] (1) mixing chloroauric acid (HAuCl4), ascorbic acid (AA), a protective agent, and water to obtain a gold nanoflower solution;

[0009] (2) mixing the gold nanoflower (HFGN) solution obtained in step (1) with a flavonoid compound solution to obtain a gold nanoflower-flavonoid compound complex solution;

[0010] (3) mixing the gold nanoflower-flavonoid compound complex solution obtained in step (2) with a biotin-PEG-SH solution to obtain the gold nanoflower-enhanced flavonoid compound.

[0011] The above method can enhance the flavonoid compound by using gold nanoflower, can enhance the photoluminescence of small molecules by plasmonic effect, greatly improves the fluorescence intensity of small molecules of traditional Chinese medicine at the tumor site, provides a more accurate and clear tumor image, and has the advantages of good targeting effect.

[0012] Preferably, the molecular weight of the flavonoid compound is 250-1000.

[0013] Preferably, the protective agent in step (1) includes any one of cetyltrimethylammonium chloride (CTAC), cetyltrimethylammonium bromide CTAC), tetraoctylammonium bromide (TOAB), oleic acid, oleylamine or lysine.

[0014] Preferably, the molar ratio of the chloroauric acid, ascorbic acid and protective agent in step (1) is 1:(150-250):(10-20), wherein the fraction of ascorbic acid can be 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250, and the fraction of the protective agent can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, but is not limited to the above-mentioned values, and other values not listed in the above-mentioned value range are also applicable.

[0015] Preferably, the flavonoid compound in step (2) includes any one of hydroxysafflor yellow A (HSYA), baicalin, quercetin, citropten, daidzein, puerarin, glycyrrhizin or glycyrrhizin.

[0016] Preferably, the volume ratio of the gold nanoflower solution to the flavonoid compound solution in step (2) is (1.5-3):1, for example 1.5:1, 2:1, 2.5:1 or 3:1, but is not limited to the above-mentioned values, and other values not listed in the above-mentioned value range are also applicable.

[0017] Preferably, the concentration of the flavonoid compound after mixing in step (2) is 0.01-20 μg / mL, and the concentration of the gold nanoflower is 0.15-0.2 mg / mL, wherein the concentration of the flavonoid compound can be 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 15 μg / mL or 20 μg / mL, etc., and the concentration of the gold nanoflower can be 0.15 mg / mL, 0.16 mg / mL, 0.17 mg / mL, 0.18 mg / mL, 0.19 mg / mL or 0.2 mg / mL, etc., but are not limited to the values ​​listed above, and other values ​​not listed within the above numerical range are also applicable.

[0018] Preferably, the concentration of the biotin-PEG-SH solution in step (3) is 30-50 mM, and the concentration of the gold nanoflower-flavonoid complex solution is 0.15-0.2 mg / mL, wherein the concentration of the biotin-PEG-SH solution can be 30 mM, 35 mM, 40 mM, 45 mM or 50 mM, etc., and the concentration of the gold nanoflower-flavonoid complex solution can be 0.15 mg / mL, 0.16 mg / mL, 0.17 mg / mL, 0.18 mg / mL, 0.19 mg / mL or 0.2 mg / mL, etc., but are not limited to the values ​​listed above, and other values ​​not listed within the above numerical range are also applicable.

[0019] Preferably, the volume ratio of the gold nanoflower-flavonoid compound complex solution to the biotin-PEG-SH solution in step (3) is (500-700):(10-15), wherein the number of parts of the gold nanoflower-flavonoid compound complex solution can be 500, 550, 600, 650 or 700, etc., and the number of parts of the biotin-PEG-SH solution can be 10, 11, 12, 13, 14 or 15, etc., but are not limited to the values ​​listed above. Other values ​​not listed within the above numerical range are also applicable.

[0020] In a second aspect, the present invention provides gold nanoflower-enhanced flavonoid compounds prepared by the preparation method described above.

[0021] In a third aspect, the present invention also provides the use of the gold nanoflowers as described above to enhance the use of flavonoid compounds in the preparation of fluorescence imaging probes.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The application provides a preparation method of gold nanoflower enhanced flavonoids, which enhances flavonoids by gold nanoflower, can enhance the photoluminescence of small molecules through plasmonic effect, greatly improves the fluorescence intensity of traditional Chinese medicine small molecules at tumor sites, provides more accurate and clear tumor images, and has the advantages of good targeting effect, high water solubility and good stability. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 UV spectra of HSYA, HFGN and HSYA-HFGN prepared in the application;

[0025] Figure 2 Fluorescence emission spectra of HSYA, HFGN and HSYA-HFGN prepared in the application;

[0026] Figure 3 Fluorescence emission spectra of HSYA and HSYA-HFGN with different concentrations prepared in the application;

[0027] Figure 4 Statistical diagram of fluorescence enhancement multiple of HSYA-HFGN with different concentrations prepared in the application;

[0028] Figure 5 Appearance of HFGN prepared in the application under a scanning electron microscope;

[0029] Figure 6 Particle size distribution diagram of HFGN prepared in the application;

[0030] Figure 7 Zeta potential result diagram of HFGN and HSYA prepared in the application;

[0031] Figure 8 In-vitro imaging diagram of HFGN, HSYA and HSYA-HFGN prepared in the application;

[0032] Figure 9 In-vivo imaging diagram of HSYA and H-H-P prepared in the application;

[0033] Figure 10 In-vivo imaging fluorescence intensity result diagram of HSYA and H-H-P prepared in the application. DETAILED DESCRIPTION

[0034] The technical solutions of the application are further illustrated by specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the application, and should not be regarded as specific limitations on the application.

[0035] In the following examples, biotin-PEG-SH was purchased from Beijing Runze Kang Biological Technology Co., Ltd., model B163356-100mg.

[0036] Example 1

[0037] This example provides a gold nanoflower enhanced flavonoid compound, and the specific steps are as follows:

[0038] (1) 10mM HAuCl4, 200mM CTAC and 300mM AA solutions were prepared with HAuCl4, AA and CTAC as raw materials and pure water as solvent. 10mL of CTAC solution was taken in a 20mL glass bottle with smooth inner wall, 1mL of HAuCl4 solution was added and mixed, 0.5mL of AA solution was added to the mixed solution, and it was fully mixed and placed in 0℃ (ice water mixture) condition. After 3h, gold nanoflower (HFGN) solution was obtained and stored in the refrigerator for use.

[0039] (2) Take 1mL of the above HFGN solution in a 5mL glass bottle, add 1mL of H2O, and add 1mL of different concentrations of hydroxyl safflower yellow A (HSYA) solution (0.01μg / mL, 0.05μg / mL, 0.1μg / mL, 0.5μg / mL, 1μg / mL, 5μg / mL, 10μg / mL, 20μg / mL) respectively, stir for 6h, and obtain gold nanoflower and small molecule complex HSYA-HFGN solution (H-H).

[0040] (3) Take all concentrations of HSYA-HFGN (H-H) solution, add 65μL of biotin-PEG-SH (DMSO as solvent, 40mM) respectively, stir for 30min, and obtain H-H-biotin-PEG-SH (H-H-P), which is the gold nanoflower enhanced flavonoid compound.

[0041] Effect test:

[0042] ① Take 3mL of HSYA solution (0.05μg / mL), HFGN solution (57μg / mL) and HSYA-HFGN solution (57μg / mL) respectively (since biotin-PEG-SH has no fluorescent reaction, the fluorescence of HSYA-HFGN can reflect the fluorescence of the product), measure and observe the absorption peak in the range of 400-1600nm with ultraviolet spectrophotometer (see Figure 1 ). Detect the fluorescence of HSYA, HFGN and HSYA-HFGN under 400nm excitation wavelength (see Figure 2 ). With the same concentration of HSYA as control, under the excitation of 400nm wavelength laser, detect the fluorescence enhancement of H-H-PEG-SH synthesized by 0.01-20μg / mL HSYA (seeFigures 3-4 It can be found from the figure that the product prepared by the scheme provided by the application can effectively enhance the fluorescence of flavonoids, is significantly better than flavonoids alone and gold nanoflowers alone, and has a fluorescence intensity better than the sum of the two.

[0043] ②10 μL of HFGN solution (57 μg / mL) was dropped on the copper mesh, and the morphology was observed by using a scanning electron microscope after being placed overnight at 20°C, as shown in Figure 5 .

[0044] ③3 mL of HFGN solution (57 μg / mL) and 3 mL of HSYA solution (0.05 μg / mL) were taken respectively, and the particle zeta potential and particle size were detected by using a particle size analyzer, and the results are shown in Figures 6-7 .

[0045] ④Hela cells were inoculated in a confocal dish at a concentration of 5×10 3 cells per well, and were placed in an incubator for 24 h to allow the cells to adhere. After 24 h, the original culture medium was discarded, and the drug 1, 0.05 μg / mL HSYA; 2, 0.05 μg / mL HSYA-HFGN; 3, HFGN with the same concentration; 4, PBS with the same volume of the drug was used to prepare the culture solution for the control group. After 12 h of drug administration, the original culture medium was aspirated, and the cells were washed with PBS for 3 times, 1 mL of PBS was added, and the CLSM was used to observe and take images (see Figure 8 ). It can be seen from the figure that the cells in the PBS group have no fluorescence, the cells in the HSYA group have almost no fluorescence, the cells in the HFGN group have strong red fluorescence, and the cells in the HSYA-HFGN group emit very strong red fluorescence, which fully shows that the product provided by the application has excellent fluorescence imaging effect at the cell level.

[0046] ⑤15 tumor-bearing mice were randomly divided into 3 groups, and were administered by tail vein (dose of administration: 150 μL): 1, the first group: 2.7 mg / mL H-H-P; 2, the second group: 2 μg / mL HSYA; 3, the third group: 50 mg / mL glucose group (Glucose). The fluorescence imaging images of the mice at 0 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h after administration were taken, and the enrichment of the drug in different tissues was detected by using a live fluorescence imaging device (excitation wavelength: 400 nm; emission wavelength: 450-700 nm wide peak) (results are shown in Figures 9-10 ). It can be found from the figure that no fluorescence was detected in the HSYA group and the glucose group at 0-24 h after administration, fluorescence was detected in the H-H-P group at 2 h, the fluorescence intensity was the strongest at 8 h, and the fluorescence intensity gradually decreased after 12 h. These contents fully show that the product provided by the application can effectively perform live imaging, and has the advantage of good targeting effect.

[0047] The applicant declares that the gold nanoflower enhanced flavonoids, the preparation method and the application thereof of the present application are illustrated by the above-mentioned embodiments, but the present application is not limited to the above-mentioned embodiments, that is, it does not mean that the present application must rely on the above-mentioned embodiments to be implemented. It should be understood by those skilled in the art that any improvement on the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes and the like all fall within the protection scope and the disclosed scope of the present application.

[0048] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above-mentioned embodiments, and various simple modifications can be made to the technical solutions of the present application within the technical concept scope of the present application, and these simple modifications all belong to the protection scope of the present application.

[0049] In addition, it should be noted that each specific technical feature described in the above-mentioned specific embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

Claims

1. A method for preparing gold nanoflower enhanced flavonoids, characterized in that, The preparation method comprises the following steps: (1) mixing chloroauric acid, ascorbic acid, a protective agent and water to obtain a gold nanoflower solution; (2) mixing the gold nanoflower solution obtained in step (1) with a flavonoid compound solution to obtain a gold nanoflower-flavonoid compound complex solution; (3) mixing the gold nanoflower-flavonoid compound complex solution obtained in step (2) with a biotin-PEG-SH solution to obtain the gold nanoflower-enhanced flavonoid compound; In step (1), the protective agent is hexadecyl trimethyl ammonium chloride; In step (1), the molar ratio of the chloroauric acid, ascorbic acid and protective agent is 1:(150-250):(10-20); In step (2), the flavonoid compound is hydroxyl safflor yellow A; In step (2), the volume ratio of the gold nanoflower solution to the flavonoid compound solution is (1.5-3):1; In step (2), the concentration of the flavonoid compound after mixing is 0.01-20 μg / mL, and the concentration of the gold nanoflower is 0.15-0.2 mg / mL.

2. The production method according to claim 1, characterized by, In step (3), the concentration of the biotin-PEG-SH solution is 30-50 mM, and the concentration of the gold nanoflower-flavonoid compound complex solution is 0.15-0.2 mg / mL.

3. The method of claim 1, wherein, In step (3), the volume ratio of the gold nanoflower-flavonoid compound complex solution to the biotin-PEG-SH solution is (500-700):(10-15).

4. A gold nanoflower-enhanced flavonoid compound prepared by the preparation method according to any one of claims 1-3.

5. Application of the gold nanoflower-enhanced flavonoid compound according to claim 4 in preparation of a fluorescence imaging probe.

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