Gold nanoflower enhanced flavonoid compound as well as preparation method and application thereof

Abstract

The invention provides a gold nanoflower enhanced flavonoid compound and a preparation method and application thereof, 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 compound solution; and (3) mixing the gold nanoflower-flavonoid compound compound 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 invention effectively enhances the fluorescence intensity of flavonoid traditional Chinese medicine micromolecules at a tumor site, provides a more accurate and clearer tumor image, and has the advantage of good targeting effect.

Description

[0001] This application claims the priority of a Chinese patent application with an application date of April 11, 2024, application number 202410436126.7, and invention name “A gold nanoflower enhanced flavonoid compound, preparation method and application thereof”, the entire contents of which are incorporated by reference into this application. 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 an application thereof, and in particular relates to a gold nanoflower-enhanced flavonoid compound with good imaging effect, a preparation method and an application thereof. Background Art

[0003] Malignant tumors (cancer) are one of the major diseases that endanger human health. The diagnosis and treatment of cancer is undoubtedly a hot topic in modern medical research. Bioluminescence imaging has shown great potential in tumor diagnosis and treatment, and its advantages include non-invasiveness, high sensitivity and high resolution. By labeling fluorescent substances or fluorescent markers, bioluminescence imaging can provide more accurate and clear tumor images, which is helpful for early diagnosis of tumors, surgical navigation and drug treatment. In the future, with the continuous development and innovation of fluorescent probe technology, the prospects for bioluminescence imaging in tumor treatment will be broader.

[0004] The active ingredients of traditional Chinese medicine have diverse structures and complex functions. Many small molecules of traditional Chinese medicine can emit fluorescence under excitation light, but the fluorescence intensity is generally weak and the luminescence efficiency is low. Therefore, how to provide a solution that can improve the fluorescence intensity of small molecules of traditional Chinese medicine while achieving excellent targeting effects has become an urgent problem to be solved. Summary of the invention

[0005] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a gold nanoflower-enhanced flavonoid compound and a preparation method and application thereof, and in particular to provide a gold nanoflower-enhanced flavonoid compound with good imaging effect and a preparation method and application thereof. The gold nanoflower-enhanced flavonoid compound provided by the present invention effectively enhances the fluorescence intensity of flavonoid Chinese medicine small molecules at the tumor site, provides a more accurate and clear tumor image, and has the advantage of good targeting effect.

[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 the 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 the biotin-PEG-SH solution to obtain the gold nanoflower-enhanced flavonoid compound.

[0011] The above method uses gold nanoflowers to enhance flavonoid compounds, which can enhance the photoluminescence of small molecules through the plasma effect, greatly improving the fluorescence intensity of traditional Chinese medicine small molecules in the tumor site, providing more accurate and clear tumor images, and has the advantage 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 hexadecyltrimethylammonium chloride (CTAC), hexadecyltrimethylammonium bromide (CTAC), tetraoctylammonium bromide (TOAB), oleic acid, oleylamine or lysine.

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

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

[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-listed values. Other values ​​not listed within the above numerical 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 compound 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 compound 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, and 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 flavonoid compounds in the preparation of fluorescent imaging probes.

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

[0023] The present invention provides a preparation method of gold nanoflower-enhanced flavonoid compounds. By using gold nanoflowers to enhance the flavonoid compounds, the photoluminescence of small molecules can be enhanced through the plasma effect, which greatly improves the fluorescence intensity of the 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, high water solubility and good stability. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0025] Figure 2 The fluorescence emission spectra of HSYA, HFGN and HSYA-HFGN prepared in the present invention;

[0026] Figure 3 The fluorescence emission spectra of HYSY and HSYA-HFGN of different concentrations prepared by the present invention are shown;

[0027] Figure 4 The fluorescence enhancement factor statistics of HSYA-HFGN with different concentrations prepared by the present invention are shown in FIG.

[0028] Figure 5 The morphology of the HFGN prepared by the present invention under a scanning electron microscope;

[0029] Figure 6 This is the particle size distribution diagram of HFGN prepared in the present invention;

[0030] Figure 7 This is a diagram showing the Zeta potential results of HFGN and HSYA prepared in the present invention;

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

[0032] Fig. 9 In vivo imaging of HSYA and HHP prepared by the present invention;

[0033] Fig.10 This is a graph showing the in vivo imaging fluorescence intensity results of HSYA and HHP prepared in the present invention. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0035] In the following examples, biotin-PEG-SH was purchased from Beijing Runzekang Biotechnology Co., Ltd., model number B163356-100mg.

[0036] Example 1

[0037] This embodiment provides a method of enhancing flavonoid compounds with gold nanoflowers, and the specific steps are as follows:

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

[0039] (2) Take 1 mL of the above HFGN solution and place it in a 5 mL glass bottle, add 1 mL of H2O, and add 1 mL of hydroxysafflor yellow A (HSYA) solution of different concentrations (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, 20 μg / mL) and stir for 6 h to obtain the gold nanoflower and small molecule complex HSYA-HFGN solution (HH).

[0040] (3) Take all the above concentrations of HSYA-HFGN (HH) solutions, add 65 μL of biotin-PEG-SH (DMSO as solvent, 40 mM) respectively, and stir for 30 minutes to obtain HH-biotin-PEG-SH (HHP), that is, the gold nanoflower enhanced flavonoid compound.

[0041] Effect test:

[0042] ① Take 3 mL of HSYA solution (0.05 μg / mL), HFGN solution (57 μg / mL) and HSYA-HFGN solution (57 μg / mL, since the biotin-PEG-SH part has no fluorescence reaction, the detection of HSYA-HFGN fluorescence can reflect the fluorescence of the product), and measure and observe the absorption peak in the range of 400-1600 nm using a UV spectrophotometer (see Figure 1 The fluorescence of HSYA, HFGN and HSYA-HFGN was detected at an excitation wavelength of 400 nm (see Figure 2 ). Using the same concentration of HSYA as a control, the fluorescence enhancement of HH-PEG-SH synthesized using 0.01-20 μg / mL HSYA was detected under laser excitation at a wavelength of 400 nm (see Figure 3-4 ). It can be found from the figure that the product prepared by the solution provided by the present invention can effectively enhance the fluorescence of flavonoid compounds, which is significantly better than the single flavonoid compounds and the single gold nanoflowers, and the fluorescence intensity is better than the sum of the two.

[0043] ② Take 10 μL of HFGN solution (57 μg / mL) and drop it on the copper grid. Place it at 20°C overnight and observe the morphology using a scanning electron microscope. Figure 5 shown.

[0044] ③ Take 3 mL of HFGN solution (57 μg / mL) and HSYA solution (0.05 μg / mL) respectively, and use a particle size analyzer to detect the particle zeta potential and particle size. The results are as follows: Figure 6-7 shown.

[0045] ④Hela cells were cultured at 5×10 3 100 cells / well were inoculated into the confocal dish and placed in the incubator for 24 hours to allow the cells to adhere to the wall. After 24 hours, the original culture medium was discarded and the complete culture medium was used to prepare drugs 1, 0.05μg / mL HSYA; 2, 0.05μg / mL HSYA-HFGN; 3, HFGN of the same concentration; 4, the control group culture medium was prepared with PBS of the same volume as the drug. 12 hours after the drug administration, the original culture medium was aspirated, washed with PBS three times, and 1mL PBS was added. The CLSM was used to observe and take images (see Figure 8 ). As can be seen from the figure, the cells in the PBS group have no fluorescence, the cells in the HYSA 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 demonstrates that the product provided by the present invention has excellent fluorescence imaging effect at the cellular level.

[0046] ⑤ 15 tumor-bearing mice were randomly divided into 3 groups and administered via tail vein (dosage 150μL): 1. Group 1: 2.7mg / mL HHP; 2. Group 2: 2μg / mL HSYA; 3. Group 3: 50mg / mL glucose group (Glucose). Fluorescence imaging was performed at 0h, 1h, 2h, 4h, 8h, 12h, and 24h after administration, and the drug enrichment in different tissues was detected using an in vivo fluorescence imaging device (excitation wavelength: 400nm; emission wavelength: 450-700nm broad peak) (see the results) Figure 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 hours after administration, while fluorescence was detected in the HHP group at 2 hours, with the strongest fluorescence intensity at 8 hours, and the fluorescence intensity gradually decreased after 12 hours. These contents fully show that the product provided by the present invention can perform effective in vivo imaging and has the advantage of good targeting effect.

[0047] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the gold nanoflower-enhanced flavonoid compounds and the preparation method and application thereof, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0048] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0049] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing gold nanoflowers to enhance flavonoid compounds, 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 the 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 the biotin-PEG-SH solution to obtain the gold nanoflower-enhanced flavonoid compound.

2. The preparation method according to claim 1, characterized in that: The protective agent in step (1) includes any one of hexadecyltrimethylammonium chloride, hexadecyltrimethylammonium bromide, tetraoctylammonium bromide, oleic acid, oleylamine or lysine.

3. The preparation method according to claim 1 or 2, characterized in that: The molar ratio of chloroauric acid, ascorbic acid and protective agent in step (1) is 1:(150-250):(10-20).

4. The preparation method according to any one of claims 1 to 3, characterized in that The flavonoid compound in step (2) includes any one of hydroxysafflor yellow A, baicalin, quercetin, nobiletin, daidzein, puerarin, isoliquiritigenin or liquiritin.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The volume ratio of the gold nanoflower solution to the flavonoid compound solution in step (2) is (1.5-3):

1.

6. The preparation method according to any one of claims 1 to 5, characterized in that: After the mixing in step (2), the concentration of flavonoid compounds is 0.01-20 μg / mL, and the concentration of gold nanoflowers is 0.15-0.2 mg / mL.

7. The preparation method according to any one of claims 1 to 6, characterized in that: The concentration of the biotin-PEG-SH solution in step (3) is 30-50 mM, and the concentration of the gold nanoflower-flavonoid compound complex solution is 0.15-0.2 mg / mL.

8. The preparation method according to any one of claims 1 to 7, characterized in that: 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).

9. A gold nanoflower enhanced flavonoid compound prepared according to the preparation method of any one of claims 1-8.

10. Use of the gold nanoflowers according to claim 9 to enhance flavonoid compounds in the preparation of fluorescence imaging probes.

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