A medical active dye and its preparation method

Abstract

The present invention discloses a method for preparing a medical active dye in the field of dye technology. The method comprises the following components: modified hydrotalcite, hydroxyl-modified Nile Red, and water. The medical active dye is obtained by loading the hydroxyl-modified Nile Red onto the modified hydrotalcite; the modified hydrotalcite is a lamellar carrier obtained by modifying the hydrotalcite with γ-aminopropyltriethoxysilane. The present invention proposes that the water solubility of the modified Nile Red is initially improved by modifying the Nile Red, and that the modified hydrotalcite is further used for loading the dye to achieve the technical effects of improved water solubility and luminescence performance.

Description

Technical Field

[0001] The invention belongs to the technical field of dyes, and in particular relates to a medical active dye and a preparation method thereof. Background Art

[0002] Medical active dyes are a class of dyes widely used in medicine and biology. They have specific biological activity and fluorescent properties and are often used in biomarkers, cell imaging, pathological diagnosis, surgical navigation, etc. Hydrotalcite is a layered double hydroxide, which is an anionic layered compound with a layered structure, interlayer ions and exchangeability. By utilizing the intercalation and interlayer ion exchangeability of the layered compound body under the action of strong polar molecules, some functional guest substances are introduced into the interlayer gaps and the layer plates are stretched apart to form a layered column compound. Nile red is a lipophilic, hydrophobic, non-polar oxazine fluorescent dye. As a medical active dye, it has a wide range of uses in the biomedical field. It has a wide range of applications and can help researchers and doctors conduct cell observation, disease diagnosis, etc. However, its poor water solubility limits its labeling of most biological molecules. Nile red does not emit light in water and will interact with water molecules to cause fluorescence quenching. At the same time, its two-photon cross-section value is too small, which makes it have certain limitations when used as a two-photon fluorescent probe to detect small biological molecules. Fluorescence quenching refers to the phenomenon that fluorescence emission is weakened or disappears through a certain mechanism. It is usually caused by the interaction of dye molecules with specific substances or the influence of environmental factors, resulting in a decrease in the fluorescence efficiency of the fluorescent substance or a shortened excited state lifetime. Fluorescence quenching mainly includes collision quenching, energy transfer, oxygen quenching, and self-quenching of fluorescent substances.

[0003] The existing technology currently has the following main problems: Nile red has poor water solubility, is easily quenched, and has a limited scope of use. Summary of the Invention

[0004] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a medical active dye and a preparation method thereof. In order to solve the problem of poor water solubility and easy quenching of Nile red, the present invention proposes to achieve a preliminary improvement in water solubility by modifying Nile red, and further use modified hydrotalcite for loading to achieve the technical effect of improving water solubility and luminescence performance.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The present invention provides a medical active dye and a preparation method thereof, wherein the medical active dye comprises the following components: modified hydrotalcite, hydroxyl-modified Nile red and water.

[0006] Preferably, the medical active dye is obtained by loading hydroxyl-modified Nile red on modified hydrotalcite.

[0007] Preferably, the modified hydrotalcite is a lamellar carrier obtained by modifying hydrotalcite with γ-aminopropyltriethoxysilane.

[0008] Preferably, the molecular formula of the hydroxyl-modified Nile Red is as follows:

[0009] .

[0010] The present invention provides a method for preparing a medical active dye, which specifically comprises the following steps:

[0011] S1. Dispersing hydrotalcite in a 10% by volume ethylene glycol solution to obtain a hydrotalcite slurry, adding γ-aminopropyltriethoxysilane, adjusting the pH to 8-10, and stirring in a water bath at 60-80° C. and 100-120 rpm for 2-3 h to obtain a modified hydrotalcite slurry;

[0012] S2, filtering, washing, and drying the modified hydrotalcite slurry obtained in S2 to obtain a modified hydrotalcite, and then dispersing the modified hydrotalcite in water to obtain a modified hydrotalcite dispersion;

[0013] S3. Mix 5-(diethylamino)-2-(nitrosophenol) and 1,6-dinaphthol to obtain a mixture, add the mixture to N,N-dimethylformamide, mix well, and heat under reflux with stirring for 3-4 hours to obtain a mixed solution;

[0014] S4, distilling the mixed solution obtained in S3 under reduced pressure to obtain a crude product, loading the sample by dry method, and subjecting the crude product to column chromatography, eluting the crude product with a mixture of ethyl acetate and petroleum ether, to separate and purify the crude product to obtain hydroxy-modified Nile red;

[0015] S5. Add the hydroxyl-modified Nile Red obtained in S4 to the modified hydrotalcite dispersion obtained in S2, and stir at 100-150 rpm for 10-15 minutes to obtain a medical active dye.

[0016] Preferably, in S1, the amount of the hydrotalcite added to the ethylene glycol solution with a volume fraction of 10% is 18-26 mg / mL.

[0017] Preferably, in S1, the added amount of γ-aminopropyltriethoxysilane is 0.6-0.8% by mass of the hydrotalcite.

[0018] Preferably, in S2, the amount of the modified hydrotalcite added to water is 10-15 mg / mL.

[0019] Preferably, in S3, the mass ratio of 5-(diethylamino)-2-(nitrosophenol) to 1,6-dinaphthol is 1:0.75-0.83.

[0020] Preferably, in S3, the amount of the mixture added to N,N-dimethylformamide is 40-50 mg / mL.

[0021] Preferably, in S4, the volume ratio of ethyl acetate to petroleum ether is 1:4-6.

[0022] Preferably, in S5, the amount of the hydroxyl-modified Nile Red added to the modified hydrotalcite dispersion is 2.5-3 ug / mL.

[0023] The beneficial effects achieved by the present invention are as follows: the present invention uses γ-aminopropyltriethoxysilane to modify hydrotalcite and loads hydroxyl-modified Nile red prepared by 5-(diethylamino)-2-(nitrosophenol) and 1,6-dinaphthol, thereby solving the problems of poor water solubility and easy quenching of Nile red; hydrotalcite is an inorganic substance, and a large number of non-bridging hydroxyl groups exist on the surface of the structure. Modifying the hydrotalcite with γ-aminopropyltriethoxysilane can not only improve the dispersibility of the hydrotalcite and generate negatively charged nanosheets when dispersed in water, but also protect the positively charged hydroxyl-modified Nile red and increase the solubility of the modified Nile red dye. and stability. The tail chain end of γ-aminopropyltriethoxysilane has a primary amine. When it is anchored near the modified Nile red, it can reduce the contact between Nile red and water and reduce the polarity. The combined effect protects the Nile red molecule from fluorescence quenching induced by the polarity of water molecules. The hydroxyl-modified Nile red prepared with 5-(diethylamino)-2-(nitrosophenol) and 1,6-dinaphthol does not change its optical properties while keeping the dye as a cationic agent. The addition of hydroxyl groups improves the water solubility of Nile red and reduces fluorescence quenching. The use of water as a solvent reduces the use of organic solvents and avoids harm to the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of the cytotoxicity test of the dyes obtained in Examples 1-3 and Comparative Examples 1-2 of the present invention are shown in FIG.

[0025] Figure 2 The graph shows the results of the oral gavage test on nude mice of the dyes obtained in Examples 1-3 and Comparative Examples 1-2 of the present invention.

[0026] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only and are not intended to limit the scope of this application.

[0029] The experimental methods in the following examples are conventional methods unless otherwise specified; the experimental materials used in the following examples are purchased from commercial channels unless otherwise specified.

[0030] Hydrotalcite (CasNo: 11097-59-9) was purchased from Beijing Yinuokai Technology Co., Ltd., catalog number H875432;

[0031] γ-Aminopropyltriethoxysilane (CasNo: 919-30-2) was purchased from Beijing Yinuokai Technology Co., Ltd., catalog number A17919;

[0032] 5-(Diethylamino)-2-(nitrosophenol) (CasNo: 25953-06-4) was purchased from Beijing Yinuokai Technology Co., Ltd., catalog number D924326;

[0033] 1,6-Dinaphthol (CasNo: 16239-18-2) was purchased from Beijing Yinuokai Technology Co., Ltd., catalog number D856259;

[0034] N,N-dimethylformamide (CasNo: 68-12-2) was purchased from Beijing Yinuokai Technology Co., Ltd., catalog number A04779;

[0035] Ethyl acetate (CasNo: 141-78-6) was purchased from Beijing Yinuokai Technology Co., Ltd., catalog number A11130;

[0036] Petroleum ether (CasNo: 8032-32-4) was purchased from Beijing Yinuokai Technology Co., Ltd., catalog number G00002.

[0037] Example 1

[0038] A method for preparing a medical active dye comprises the following steps:

[0039] S1. Dispersing hydrotalcite at an addition amount of 18 mg / mL into a 10% by volume ethylene glycol solution to obtain a hydrotalcite slurry, adding γ-aminopropyltriethoxysilane (0.6% by mass of the hydrotalcite), adjusting the pH to 8, and stirring at 100 rpm in a 60°C water bath for 2 h to obtain a modified hydrotalcite slurry;

[0040] S2, filtering, washing, and drying the modified hydrotalcite slurry obtained in S2 to obtain a modified hydrotalcite, and then dispersing the modified hydrotalcite in water at an addition amount of 10 mg / mL to obtain a modified hydrotalcite dispersion;

[0041] S3. 5-(diethylamino)-2-(nitrosophenol) and 1,6-dinaphthol were mixed in a mass ratio of 1:0.75 to obtain a mixture, and the mixture was added to N,N-dimethylformamide in an amount of 40 mg / mL. The mixture was mixed evenly, and the mixture was stirred, heated, and refluxed for 3 h to obtain a mixed solution.

[0042] The reaction process for preparing the hydroxyl-modified Nile red is as follows:

[0043] ;

[0044] S4, distilling the mixed solution obtained in S3 under reduced pressure to obtain a crude product, dry-loading the sample, and subjecting the crude product to column chromatography, eluting with a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:4, to separation and purification to obtain hydroxy-modified Nile red;

[0045] S5. Add the hydroxy-modified Nile red obtained in S4 at an amount of 2.5 ug / mL to the modified hydrotalcite dispersion obtained in S2, and stir at 100 rpm for 10 min to obtain a medical active dye.

[0046] Example 2

[0047] A method for preparing a medical active dye comprises the following steps:

[0048] S1. Dispersing 26 mg / mL of hydrotalcite into a 10% by volume ethylene glycol solution to obtain a hydrotalcite slurry, adding 0.8% by mass of γ-aminopropyltriethoxysilane to the hydrotalcite, adjusting the pH to 10, and stirring at 120 rpm in an 80°C water bath for 3 h to obtain a modified hydrotalcite slurry;

[0049] S2, filtering, washing, and drying the modified hydrotalcite slurry obtained in S2 to obtain a modified hydrotalcite, and then dispersing the modified hydrotalcite into water at an addition amount of 15 mg / mL to obtain a modified hydrotalcite dispersion;

[0050] S3. 5-(diethylamino)-2-(nitrosophenol) and 1,6-dinaphthol were mixed in a mass ratio of 1:0.83 to obtain a mixture, and the mixture was added to N,N-dimethylformamide in an amount of 50 mg / mL. The mixture was mixed evenly, and heated under reflux with stirring for 4 h to obtain a mixed solution.

[0051] S4, distilling the mixed solution obtained in S3 under reduced pressure to obtain a crude product, dry-loading the sample, and subjecting the crude product to column chromatography, eluting with a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:6, to separate and purify the crude product to obtain hydroxy-modified Nile red;

[0052] S5. Add the hydroxy-modified Nile red obtained in S4 at an amount of 3 ug / mL to the modified hydrotalcite dispersion obtained in S2, and stir at 150 rpm for 15 minutes to obtain a medical active dye.

[0053] Example 3

[0054] A method for preparing a medical active dye comprises the following steps:

[0055] S1. Dispersing 22 mg / mL of hydrotalcite into a 10% by volume ethylene glycol solution to obtain a hydrotalcite slurry, adding 0.7% by mass of γ-aminopropyltriethoxysilane to the hydrotalcite, adjusting the pH to 9, and stirring in a 70°C water bath at 110 rpm for 2.5 h to obtain a modified hydrotalcite slurry;

[0056] S2, filtering, washing, and drying the modified hydrotalcite slurry obtained in S2 to obtain a modified hydrotalcite, and then dispersing the modified hydrotalcite in water at an addition amount of 12.5 mg / mL to obtain a modified hydrotalcite dispersion;

[0057] S3. 5-(diethylamino)-2-(nitrosophenol) and 1,6-dinaphthol were mixed in a mass ratio of 1:0.79 to obtain a mixture, and the mixture was added to N,N-dimethylformamide in an amount of 45 mg / mL. The mixture was mixed evenly, and the mixture was stirred, heated, and refluxed for 3.5 h to obtain a mixed solution.

[0058] S4, distilling the mixed solution obtained in S3 under reduced pressure to obtain a crude product, dry-loading the sample, and subjecting the crude product to column chromatography, eluting with a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:5, to separate and purify the crude product to obtain hydroxy-modified Nile red;

[0059] S5. Add the hydroxy-modified Nile red obtained in S4 at an amount of 2.8 ug / mL to the modified hydrotalcite dispersion obtained in S2, and stir at 130 rpm for 12 min to obtain a medical active dye.

[0060] Comparative Example 1

[0061] S1. Dispersing hydrotalcite in water at an addition amount of 10 mg / mL to obtain a hydrotalcite dispersion;

[0062] S2. 5-(diethylamino)-2-(nitrosophenol) and 1,6-dinaphthol were mixed in a mass ratio of 1:0.75 to obtain a mixture, and the mixture was added to N,N-dimethylformamide in an amount of 40 mg / mL. The mixture was mixed evenly, and heated under reflux with stirring for 3 h to obtain a mixed solution;

[0063] S3, distilling the mixed solution obtained in S2 under reduced pressure to obtain a crude product, dry-loading the sample, and subjecting the crude product to column chromatography, eluting with a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:4, to separate and purify the crude product to obtain hydroxy-modified Nile red;

[0064] S4. Add the hydroxy-modified Nile red obtained in S3 at an amount of 2.5 ug / mL to the hydrotalcite dispersion obtained in S2, and stir at 100 rpm for 10 minutes to obtain a medical active dye.

[0065] Comparative Example 2

[0066] S1. Dispersing hydrotalcite at an addition amount of 18 mg / mL into a 10% by volume ethylene glycol solution to obtain a hydrotalcite slurry, adding γ-aminopropyltriethoxysilane (0.6% by mass of the hydrotalcite), adjusting the pH to 8, and stirring at 100 rpm in a 60°C water bath for 2 h to obtain a modified hydrotalcite slurry;

[0067] S2, filtering, washing, and drying the modified hydrotalcite slurry obtained in S2 to obtain a modified hydrotalcite, and then dispersing the modified hydrotalcite in water at an addition amount of 10 mg / mL to obtain a modified hydrotalcite dispersion;

[0068] S3. Add 2.5 ug / mL of Nile Red to the modified hydrotalcite dispersion obtained in S2, and stir at 100 rpm for 10 min to obtain a medical active dye.

[0069] Experimental Example 1 Cytotoxicity Test

[0070] The adipocyte concentration was adjusted to 1 × 10 5 / mL, suspended with basic culture medium and inoculated into 96-well plates, 100uL of adipocyte suspension was added to each well and cultured until the cells adhered to the wall, washed twice with PBS, 100uL of Example 1-3 and Comparative Example 1-2 were added to each well, and 100uL of basic culture medium was added to the control group. After culturing for 24h, CCK-8 was added, 10uL was taken from each well, and the absorbance at 450nm was measured after incubation for 48h. The survival rate was calculated using the following formula.

[0071] The formula is: survival rate = absorbance value of experimental group / absorbance value of control group × 100%.

[0072] Figure 1 This is a graph showing the results of the cytotoxicity test of the dyes obtained in Examples 1-3 and Comparative Examples 1-2 of the present invention. As shown in the figure, the survival rates of the control group, Examples 1-3, and Comparative Examples 1-2 are 106.6%, 92.9%, 91.3%, 93.3%, 89.1%, and 86.5%, respectively; it can be seen that the 24-hour survival rate of the cells is higher than 85%. The toxicity of Examples 1-3 and Comparative Examples 1-2 on fat cells was investigated using the CCK-8 method, which indicates that Examples 1-3 and Comparative Examples 1-2 have lower cytotoxicity and good biocompatibility.

[0073] Experimental Example 2 Nude Mouse Gavage Test

[0074] Three nude mice were taken in each group. The control group was gavage-administered with an aqueous suspension of Nile red, and the experimental group was gavage-administered with an equivalent molar amount of modified Nile red of Examples 1-3 and Comparative Examples 1-2. Three hours after administration, the mice were placed in a living imaging device for imaging and fluorescence intensity was counted. The imaging mode was fluorescence mode, the excitation wavelength was 550 nm, the emission wavelength was 620 nm, and the exposure time was 0.1 s. The fluorescence intensity growth rate was calculated using the following formula:

[0075] The formula is: Growth rate = (fluorescence intensity of the experimental group - fluorescence intensity of the control group) / fluorescence intensity of the control group × 100%

[0076] Figure 2 This is a graph showing the results of the nude mouse gavage test of the dyes obtained in Examples 1-3 of the present invention and Comparative Example 1-2. As shown in the figure, the growth rates of Examples 1-3 and Comparative Example 1-2 are 326.2%, 331.9%, 327.5%, 126.3%, and 156.8%, respectively; the growth rates of Examples 1-3 and Comparative Example 1-2 are all positive, and Example 1-3 is significantly greater than Comparative Example 1-2, indicating that the modification of hydrotalcite and Nile red enhances the luminescent properties of the fluorescent dye.

[0077] While the embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that various changes, modifications, substitutions, and alterations can be made to the embodiments without departing from the principles and spirit of the invention.

[0078] The present invention and its embodiments are described above. Such description is not restrictive. The drawings show only one embodiment of the present invention, and actual applications are not limited thereto. In short, if a person skilled in the art is inspired by the above, and does not deviate from the purpose of the present invention, any method and embodiment similar to the technical solution without creative design shall fall within the scope of protection of the present invention.

Claims

1. A medical active dye, characterized in that: The invention comprises the following components: modified hydrotalcite, hydroxyl-modified Nile red and water; the medical active dye is obtained by loading hydroxyl-modified Nile red on modified hydrotalcite, and the modified hydrotalcite is a lamellar carrier obtained by modifying hydrotalcite with γ-aminopropyltriethoxysilane; The preparation method of the medical active dye specifically comprises the following steps: S1. Dispersing hydrotalcite in a 10% by volume ethylene glycol solution to obtain a hydrotalcite slurry, adding γ-aminopropyltriethoxysilane, adjusting the pH to 8-10, and stirring in a water bath at 60-80° C. and 100-120 rpm for 2-3 h to obtain a modified hydrotalcite slurry; S2, filtering, washing, and drying the modified hydrotalcite slurry obtained in S2 to obtain a modified hydrotalcite, and then dispersing the modified hydrotalcite in water to obtain a modified hydrotalcite dispersion; S3, mixing 5-diethylamino-2-nitrosophenol and 1,6-dinaphthol to obtain a mixture, adding the mixture to N,N-dimethylformamide, mixing evenly, and stirring and heating under reflux for 3-4 hours to obtain a mixed solution; S4, distilling the mixed solution obtained in S3 under reduced pressure to obtain a crude product, loading the sample by dry method, and subjecting the crude product to column chromatography, eluting the crude product with a mixture of ethyl acetate and petroleum ether, to separate and purify the crude product to obtain hydroxy-modified Nile red; S5. Add the hydroxyl-modified Nile Red obtained in S4 to the modified hydrotalcite dispersion obtained in S2, and stir at 100-150 rpm for 10-15 minutes to obtain a medical active dye.

2. The medical active dye according to claim 1, wherein: In S1, the amount of the hydrotalcite added to the ethylene glycol solution with a volume fraction of 10% is 18-26 mg / mL.

3. The medical active dye according to claim 2, wherein: In S1, the added amount of the γ-aminopropyltriethoxysilane is 0.6-0.8% by mass of the hydrotalcite.

4. The medical active dye according to claim 3, wherein: In S2, the modified hydrotalcite is added to water in an amount of 10-15 mg / mL.

5. The medical active dye according to claim 4, wherein: In S3, the mass ratio of 5-diethylamino-2-nitrosophenol to 1,6-dinaphthol is 1:0.75-0.

83.

6. The medical active dye according to claim 5, characterized in that: In S3, the mixture is added to N,N-dimethylformamide in an amount of 40-50 mg / mL.

7. The medical active dye according to claim 6, wherein: In S4, the volume ratio of ethyl acetate to petroleum ether is 1:4-6.

8. The medical active dye according to claim 7, wherein: In S5, the amount of the hydroxyl-modified Nile Red added to the modified hydrotalcite dispersion is 2.5-3 ug / mL.

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