Curcumin quantum dot as well as preparation method and application thereof

Abstract

The invention relates to a curcumin quantum dot, a preparation method thereof and application of the curcumin quantum dot in preparation of a tumor diagnosis and treatment integrated reagent. The curcumin quantum dot comprises a reaction product formed by performing a Schiff base reaction on curcumin and a polyamino ligand in the presence of a dehydrating agent, wherein the average diameter of the curcumin quantum dot is 1-30nm. The curcumin quantum dot provided by the invention can greatly improve the solubility, biocompatibility and antitumor activity of single curcumin.

Description

Technical Field

[0001] The invention relates to the field of medical materials, and in particular to curcumin quantum dots, a preparation method thereof and use thereof. Background Art

[0002] As a traditional treasure of my country, traditional Chinese medicine based on traditional Chinese medicine has shown excellent therapeutic effects as an adjuvant treatment after surgery, chemotherapy, radiotherapy or other types of treatment. Reports show that traditional Chinese medicine can improve the survival rate and quality of life of patients undergoing surgery and chemotherapy, reduce adverse reactions to chemotherapy, and overcome chemotherapy resistance; combined with radiotherapy can achieve radiosensitization and reduce side effects; in addition, it can also provide potential treatment options for combined immune therapy by regulating the tumor microenvironment. Traditional Chinese medicine can exert its anti-tumor effect by regulating oncogenes and tumor suppressor genes, epigenetic modifications, microenvironment and tumor stem cells. In fact, many modern drugs are extracts and derivatives of traditional Chinese herbal medicines and natural products. Natural products can affect multiple pathways including cell apoptosis, proliferation, invasion / migration, angiogenesis and metastasis to exert anti-tumor effects. Due to their chemical diversity, multi-target potential, low toxicity, safety and accessibility, they are more attractive than synthetic drugs.

[0003] Curcumin is a phenolic compound extracted from turmeric plants. It has natural anticancer, anti-inflammatory and antioxidant pharmacological activities. It has shown promising chemoprevention and anticancer activities in different cancer models, such as prostate cancer, lung cancer, breast cancer, brain tumors, head and neck squamous cell carcinoma, etc. The anticancer activity of curcumin mainly includes inducing cell apoptosis and inhibiting multiple cell signaling pathways to inhibit tumor proliferation and invasion. Many studies at home and abroad have shown that in the occurrence and development of tumors, curcumin can increase the level of TAZ ubiquitination, significantly inhibit the self-repair of circulating tumor stem cells, and limit their metastasis. At the same time, it inhibits cancer cell proliferation, migration and invasion through gene regulation and multiple signaling pathways. However, due to the close relationship between molecular structure and inherent physicochemical properties, curcumin has poor water solubility, which seriously affects oral bioavailability and chemical stability. The hydrophobic properties of curcumin reduce the cellular uptake rate and poor absorption, which leads to low utilization of curcumin in cells. In addition, curcumin has a fast metabolism and is quickly eliminated throughout the body. The minimum oral dose that can cause detectable levels of curcumin in plasma is high, and human studies have shown that curcumin alone, at low doses, is undetectable in plasma.

[0004] In response to the above problems, we conducted a design and preparation strategy for curcumin-based quantum dots. Quantum dots have good biocompatibility, photostability and unique luminescence properties, and the emission wavelength can be adjusted as needed. They have been widely used in biological detection of bacteria, tumors, etc. We used materials and chemical synthesis methods, took curcumin as a precursor, and designed and synthesized curcumin Chinese medicine quantum dots through carbonyl-amine condensation, Schiff base reaction, etc., combined with traditional Chinese medicine decoction process. The size effect of quantum dots is used to improve the solubility and biocompatibility of curcumin, compound multiple compounds, and improve the efficacy of curcumin compound drugs, while giving play to the fluorescence characteristics of quantum dots. The traditional Chinese medicine material curcumin is endowed with new functions such as solubility, biocompatibility, and fluorescence characteristics of quantum dots, and the application value of curcumin in tumor diagnosis and treatment is optimized. The fluorescence and controllable construction characteristics of quantum dots are used to achieve combined diagnosis and treatment and multifunctionality. On the one hand, the interdisciplinary characteristics are brought into play, and the experience advantages of traditional Chinese medicine are used to provide guidance for drug development. On the other hand, it also empowers traditional medical materials and expands application scenarios, efficacy, functions, etc. with the help of materials chemistry and other means. Summary of the invention

[0005] In response to the above problems, the applicant provides a new curcumin quantum dot design and preparation strategy. In particular, the technical problem to be solved by the present invention is to design and synthesize quantum dot materials based on the traditional Chinese medicine curcumin, and utilize the size effect of quantum dots to improve the solubility and biocompatibility of curcumin; by compounding the curcumin quantum dot materials with a variety of anti-tumor compounds, the efficacy of the curcumin compound drug in anti-tumor cells is improved; and at the same time, the fluorescence characteristics of quantum dots are utilized to achieve staining and marking of tumor cells.

[0006] In order to solve the above technical problems, this application adopts the following technical solutions:

[0007] A curcumin quantum dot comprises a reaction product formed by a Schiff base reaction of curcumin and a polyamino ligand in the presence of a dehydrating agent, wherein the average diameter of the curcumin quantum dot is 1-30 nm, preferably 3-8 nm, and more preferably 3-5 nm.

[0008] Optionally, the number of amino groups in the polyamino ligand is equal to or greater than 2.

[0009] Optionally, the polyamino ligand is selected from one of ethylenediamine, p-phenylenediamine, o-phenylenediamine, benzene-1,2,3-triamine, triaminoguanidine hydrochloride and 2,4,6-triaminopyrimidine or any combination thereof, preferably ethylenediamine.

[0010] Optionally, the dehydrating agent is selected from one or more of DCC and concentrated sulfuric acid.

[0011] Optionally, the reaction product is a Schiff base polymer formed by a Schiff base reaction between two carbonyl groups of curcumin and multiple amino groups of the polyamino ligand. The weight average molecular weight of the Schiff base polymer may be 900-2000 Da.

[0012] A method for preparing curcumin quantum dots comprises the following steps:

[0013] S1-providing a solution of curcumin in a reaction solvent;

[0014] S2-reacting curcumin with polyamino ligands in the solution for a period of time; then

[0015] S3-adding a dehydrating agent to continue the reaction, thereby obtaining a solution of the reaction product; and

[0016] S4-The reaction product is purified and dried to obtain curcumin quantum dots.

[0017] Optionally, the reaction solvent is selected from one or more of ultrapure water, ethanol, N,N-dimethylacetamide (DMAC), N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO).

[0018] Optionally, the reaction temperature of step S2 is 25-210° C., the reaction time is 10-20 hours, and the reaction process includes hydrothermal reaction or reflux reaction.

[0019] Optionally, the reaction temperature of step S3 is 25-210° C., and the reaction time is 1-10 hours.

[0020] Optionally, the purification in step S4 includes filtering to remove the dehydrating agent, removing excess solvent and washing with water.

[0021] Optionally, the drying of step S4 includes freeze drying or heat drying, the freeze drying temperature is -200 to -4°C, and the heat drying temperature is 25 to 100°C.

[0022] Optionally, the weight ratio of curcumin to amino ligand is (10-90):(10-50).

[0023] The curcumin quantum dots are used in the preparation of an integrated diagnosis and treatment reagent for tumors, wherein the integrated diagnosis and treatment includes staining and marking of tumor cells and therapeutic inhibition of tumor cells.

[0024] The beneficial effects of the present invention are:

[0025] 1) The curcumin quantum dots of the present invention can significantly improve the solubility, biocompatibility and / or anti-tumor activity of single curcumin, for example, causing tumor cell apoptosis, and resisting tumor cell migration and invasion.

[0026] 2) The curcumin quantum dots of the present invention can be compounded with a variety of anti-tumor compounds to enhance the efficacy of the curcumin compound drug in anti-tumor cells.

[0027] 3) The curcumin quantum dots of the present invention have the fluorescence characteristics of quantum dots and can stain and mark tumor cells to achieve integrated diagnosis and treatment.

[0028] 4) The preparation of curcumin quantum dots described in the present invention can enable traditional medical materials to be empowered, and by means of materials chemistry and other means, the application scenarios, therapeutic effects, functions, etc. can be expanded. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a comparison chart of the solubility in pure water of the curcumin (No. 0) of Comparative Example 1 and the curcumin quantum dots prepared in Examples 1-4 (corresponding to No. 1, No. 2, No. 3 and No. 4, respectively).

[0030] Figure 2 A, B, C and D are transmission electron micrographs of curcumin quantum dots prepared in Example 1, Example 2, Example 3 and Example 4, respectively.

[0031] Figure 3 The Fourier transform infrared (FTIR) spectrum (left) and ultraviolet-visible (UV-vis) analysis spectrum (right) of the curcumin quantum dots prepared in Example 1.

[0032] Figure 4 The Fourier transform infrared (FTIR) spectrum (left) and ultraviolet-visible (UV-vis) analysis spectrum (right) of the curcumin quantum dots prepared in Example 2.

[0033] Figure 5 The Fourier transform infrared (FTIR) spectrum (left) and ultraviolet-visible (UV-vis) analysis spectrum (right) of the curcumin quantum dots prepared in Example 3.

[0034] Figure 6 The Fourier transform infrared (FTIR) spectrum (left) and ultraviolet-visible (UV-vis) analysis spectrum (right) of the curcumin quantum dots prepared in Example 4.

[0035] Figure 7 It is a graph showing the CCK8 experimental results of pure curcumin of Comparative Example 1 and four kinds of curcumin quantum dots prepared in Examples 1-4 (1: Example 1; 2: Example 2; 3: Example 3 and 4: Example 4).

[0036] Figure 8It is a graph showing the apoptosis experimental results of pure curcumin of Comparative Example 1 and four kinds of curcumin quantum dots prepared in Examples 1-4 (1: Example 1; 2: Example 2; 3: Example 3 and 4: Example 4).

[0037] Fig. 9 It is a graph showing the migration experiment results of pure curcumin of Comparative Example 1 and four kinds of curcumin quantum dots prepared in Examples 1-4 (1: Example 1; 2: Example 2; 3: Example 3 and 4: Example 4).

[0038] Fig.10 It is a graph showing the results of invasion experiments of pure curcumin of Comparative Example 1 and four kinds of curcumin quantum dots prepared in Examples 1-4 (1: Example 1; 2: Example 2; 3: Example 3 and 4: Example 4).

[0039] Fig.11 These are fluorescent confocal photographs of cells stained with four kinds of curcumin quantum dots prepared in Examples 1-4 (1: Example 1; 2: Example 2; 3: Example 3 and 4: Example 4). DETAILED DESCRIPTION

[0040] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this area or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0041] In this article, the term "quantum dots" refers to tiny crystalline particles that often produce quantum effects, and the particle size is usually a few nanometers to tens of nanometers.

[0042] As used herein, the term "polyamino ligand" refers to a ligand compound containing two or more amino functional groups.

[0043] In this article, the term "Schiff base reaction" refers to the condensation reaction of carbonyl compounds and ammonia compounds under appropriate conditions through carbonyl-amine condensation. It has a wide range of applications in organic synthesis and drug development. It can be used to synthesize various compounds, such as amides, ketoamines, imines, etc. These compounds have important applications in the fields of medicine and pesticides. Carbonyl-amine condensation reactions are also widely used in the synthesis of key intermediates or active parts in drugs, thereby improving synthesis efficiency and product purity. Carbonyl-amine condensation reactions are used in the synthesis of many anticancer drugs and antibacterial drugs. In addition, carbonyl-amine condensation reactions also have important applications in biochemical research. In the study of post-translational modifications of proteins, drug target identification, etc., carbonyl-amine condensation reactions are needed to synthesize specific compounds.

[0044] The present invention relates to the field of pharmaceutical materials, in particular to the field of Chinese medicine processing and synthesis, and in particular to a quantum dot-type Chinese medicine material prepared based on curcumin. The present invention also discloses a method for preparing a new quantum dot material based on the traditional Chinese medicine material curcumin through a condensation reaction, as well as the efficacy of curcumin quantum dots in the field of cell staining and anti-tumor. The curcumin-based quantum dot material disclosed in the present invention and its preparation method can effectively improve the bioavailability and water solubility of traditional curcumin monomers, enhance its efficacy and reduce its toxicity to normal cells, while giving it new functions such as fluorescence characteristics and cell staining, and is expected to achieve the combined diagnosis and treatment of tumors. On the one hand, it gives play to the characteristics of interdisciplinary studies and provides guidance for drug development with the experience advantages of traditional Chinese medicine. On the other hand, it also empowers traditional medical materials, and expands application scenarios, efficacy, functions, etc. with the help of materials science chemistry and other means.

[0045] Quantum dots have good biocompatibility, photostability and unique luminescence properties, and the emission wavelength can be adjusted as needed. They have been widely used in biological detection of bacteria, tumors, etc. The applicant used materials and chemical synthesis methods, took curcumin as a precursor, and designed and synthesized curcumin Chinese medicine quantum dots through carbonylamine condensation, Schiff base reaction, etc., combined with traditional Chinese medicine decoction technology. The size effect of quantum dots is used to improve the solubility and biocompatibility of curcumin, compound a variety of anti-tumor compounds, improve the efficacy of curcumin compound drugs, and at the same time give play to the fluorescence characteristics of quantum dots. The traditional Chinese medicine material curcumin is given new functions such as solubility, biocompatibility, and fluorescence characteristics of quantum dots, optimizing the application value of curcumin in both tumor diagnosis and treatment. The fluorescence and controllable construction characteristics of quantum dots are used to achieve combined diagnosis and treatment and multifunctionality.

[0046] Therefore, in one aspect, the present invention provides a curcumin quantum dot, comprising a reaction product formed by a Schiff base reaction of curcumin and a polyamino ligand in the presence of a dehydrating agent, wherein the reaction product comprises a -C=N- group and the average diameter of the curcumin quantum dot is 1-30 nm.

[0047] As a preferred embodiment of the curcumin quantum dots of the present invention, the average diameter of the curcumin quantum dots is 3-8 nm, more preferably 3-5 nm.

[0048] As a preferred embodiment of the curcumin quantum dots of the present invention, the number of amino groups in the polyamino ligand is equal to or greater than 2.

[0049] The reaction product can be a Schiff base polymer formed by the two carbonyl groups of curcumin and the multiple amino groups of the polyamino ligand through the Schiff base reaction. The weight average molecular weight of the Schiff base polymer can be 900-2000Da as measured by GPC. As a preferred embodiment of the curcumin quantum dots of the present invention, the polyamino ligand is selected from one of ethylenediamine, p-phenylenediamine, o-phenylenediamine, benzene-1,2,3-triamine, triaminoguanidine hydrochloride and 2,4,6-triaminopyrimidine or any combination thereof, more preferably ethylenediamine.

[0050] As a preferred embodiment of the curcumin quantum dots of the present invention, the dehydrating agent is selected from one or more of DCC and concentrated sulfuric acid.

[0051] As a preferred embodiment of the curcumin quantum dots of the present invention, the reaction product is a branched polymer formed by the two carbonyl groups of curcumin and the multiple amino groups of the polyamino ligand through a Schiff base reaction.

[0052] In a second aspect, the present invention provides a method for preparing curcumin quantum dots, comprising the following steps:

[0053] S1-providing a solution of curcumin in a reaction solvent;

[0054] S2-reacting curcumin with polyamino ligands in the solution for a period of time; then

[0055] S3-adding a dehydrating agent to continue the reaction, thereby obtaining a solution of the reaction product; and

[0056] S4-The reaction product is purified and dried to obtain curcumin quantum dots.

[0057] As a preferred solution for preparing the curcumin quantum dots of the present invention, the reaction solvent is selected from one or more of ultrapure water, ethanol, N,N-dimethylacetamide (DMAC), N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO).

[0058] As a preferred solution for preparing curcumin quantum dots according to the present invention, the reaction temperature of step S2 is 25-210° C., the reaction time is 10-20 hours, and the reaction process includes hydrothermal reaction or reflux reaction.

[0059] As a preferred solution for preparing curcumin quantum dots according to the present invention, the reaction temperature of step S3 is 25-210° C., and the reaction time is 1-10 hours.

[0060] As a preferred solution for preparing the curcumin quantum dots of the present invention, the purification in step S4 includes filtering to remove the dehydrating agent, removing the excess solvent and washing with water.

[0061] As a preferred solution for preparing curcumin quantum dots according to the present invention, the drying in step S4 includes freeze drying or heat drying, the freeze drying temperature is -200 to -4°C, and the heat drying temperature is 25 to 100°C.

[0062] As a preferred solution for preparing the curcumin quantum dots of the present invention, the weight ratio of curcumin to amino ligand is (10-90):(10-50).

[0063] In a third aspect, the present invention also provides the use of curcumin quantum dots in the preparation of an integrated diagnosis and treatment reagent for tumors, wherein the integrated diagnosis and treatment includes staining and marking of tumor cells and therapeutic inhibition of tumor cells.

[0064] Preferably, the curcumin quantum dots of the present invention have tumor cell staining function.

[0065] Preferably, the curcumin quantum dots of the present invention have anti-tumor cell efficacy.

[0066] The present invention also relates to a method for staining tumor cells using curcumin quantum dots, comprising the following steps:

[0067] Cells in the logarithmic growth phase were digested with trypsin to obtain a cell suspension, which was diluted with complete culture medium and added to a 6-well plate. After the cells adhered to the wall, complete culture medium was added and incubated. The culture medium was discarded, and curcumin and curcumin quantum dots were added. The cells were washed after incubation and staining, and the cell morphology and fluorescence imaging were observed using a laser confocal microscope system.

[0068] The present invention also relates to a method for detecting the anti-tumor cell efficacy of curcumin quantum dots, comprising the following steps:

[0069] 1. Cytotoxicity detection: CCK-8 kit was used to determine the toxicity of curcumin and curcumin quantum dots to human lung non-small cell carcinoma A549 cells. The cell suspension was added to a 96-well plate and incubated in a cell culture incubator at 37°C and 5% CO2. Curcumin and curcumin quantum dot solutions prepared with sterile deionized water of different mass concentrations were added to each well. After continued incubation, CCK-8 reagent was added to determine the cell survival rate and calculate the IC50 value.

[0070] 2. Cell apoptosis detection: A549 cell suspension was inoculated on a 6-well plate and incubated in a cell culture incubator at 37°C and 5% CO2. Curcumin and curcumin quantum dots were added. After serum-free incubation, serum was added for incubation. The culture medium was discarded. After washing, the cell suspension was prepared by trypsin digestion. After staining with live and dead dyes, the cell suspension was incubated on ice in the dark. After washing and resuspending, the cell suspension was blocked. After antibody incubation, the cell suspension was washed and resuspended for loading on a flow cytometer.

[0071] 3. Scratch experiment: A549 cells were seeded in a 6-well plate overnight, and the Tip head artificially made scratches of similar size in the 6-well plate. After washing, 40μM curcumin and curcumin quantum dots were added for treatment. After 24 hours, the scratch width was observed and measured, and the average scratch width was obtained by measuring three random areas, and the migration distance and cell migration rate were calculated.

[0072] 4. Transwell invasion assay: A549 cells were starved for 12 hours after serum withdrawal to prepare cell suspension, 1000 A549 cells were counted and treated with curcumin and curcumin quantum dot culture medium respectively. Added to Transwell microporous filter culture chamber, culture medium containing 15% FBS was added to the lower chamber of 24-well plate, the chamber was taken out after incubation, the culture medium was discarded and washed with DPBS, the residual cells on the upper layer of the chamber were wiped off with cotton swabs, ethanol fixed, crystal violet stained, and counted under a microscope.

[0073] example

[0074] The invention is further described in conjunction with the following specific examples. It should be understood that the specific examples described herein are only used to explain the present invention and are not intended to limit the present invention. The various raw materials and reagents used in the present invention are conventional commercial reagents and can be directly obtained by purchasing from the market.

[0075] DMAC: abbreviation for dimethylacetamide

[0076] DMSO: abbreviation for dimethyl sulfoxide

[0077] The structural formula of curcumin is:

[0078] The structural formula of ethylenediamine is:

[0079] The structural formula of 2,4,6-triaminopyrimidine is

[0080] The structural formula of phenylenediamine is

[0081] The structural formula of triaminoguanidine hydrochloride

[0082] DCC: abbreviation of N,N-dicyclohexylcarbodiimide

[0083] Transmission electron microscope model JEM-2100, obtained from JEOL Ltd. Fourier transform infrared spectrometer model Bruker INVENIO S, obtained from Bruker Corporation

[0084] Curcumin, obtained from Guangdong Chemical Co., Ltd., with a purity of 99.90%.

[0085] Example 1 Curcumin quantum dots prepared based on ethylenediamine

[0086] Use a mixed solution of pure water and DMAC as solvent (volume ratio 1:1), add curcumin, stir at 30°C for 30 minutes, and then ultrasonicate for 30 minutes to make it evenly dissolved. Then add ethylenediamine at a molar ratio of 1:1 to curcumin, reflux at 180°C for 12 hours, add DCC dehydrating agent and continue stirring for 5 hours.

[0087] After filtering out the dehydrating agent, the excess solvent was removed by vacuum rotary evaporation, and the insoluble matter was removed by soaking in pure water, followed by freeze drying (vacuum, -40 degrees, freezing time 12h) to obtain the curcumin quantum dots (No. 1) of Example 1. The weight average molecular weight of the curcumin quantum dots measured by gel permeation chromatography (GPC) was 910Da, and GPC was measured at room temperature on a Tosoh High-TemperatureEcoSEC detector using 1,2,4-trichlorobenzene as an eluent.

[0088] Example 2 Curcumin quantum dots prepared based on 2,4,6-triaminopyrimidine

[0089] A mixed solution of pure water and DMSO was used as a solvent (volume ratio of 1:1.5), and curcumin was added, stirred at 30°C for 30 minutes, and then ultrasonicated for 30 minutes to make it uniformly dissolved. Then 2,4,6-triaminopyrimidine was added at a molar ratio of 2:3 to curcumin, and the mixture was reacted in a hydrothermal reactor at 150°C for 12 hours.

[0090] The excess solvent was removed by vacuum rotary evaporation, and the insoluble matter and unreacted ligand were removed by soaking in pure water, followed by freeze drying (vacuum, -40 degrees, freezing time 13 hours) to obtain the curcumin quantum dots (No. 2) of Example 2. The weight average molecular weight of the curcumin quantum dots measured by gel permeation chromatography (GPC) was 1220 Da, and GPC was measured at room temperature on a Tosoh High-Temperature EcoSEC detector using 1,2,4-trichlorobenzene as an eluent.

[0091] Example 3 Curcumin quantum dots prepared based on p-phenylenediamine

[0092] Use pure water and DMAC mixed solution as solvent (volume ratio 1:1), add curcumin, stir at 30°C for 30 minutes, then ultrasonic for 30 minutes to make it evenly dissolved. Then add phenylenediamine, with a molar ratio of 1:1 to curcumin, reflux at 180°C for 12 hours, add concentrated sulfuric acid dehydrating agent and continue stirring for 5 hours.

[0093] After soaking in pure water to remove insoluble matter, the solid phase product was separated by vacuum rotary evaporation, and then repeatedly soaked and washed with pure water, and the final product was dried in an oven at 60°C to obtain the curcumin quantum dots (No. 3) of Example 3. The weight average molecular weight of the curcumin quantum dots measured by gel permeation chromatography (GPC) was 1001 Da, and GPC was measured on a Tosoh High-TemperatureEcoSEC detector at room temperature using 1,2,4-trichlorobenzene as an eluent.

[0094] Example 4 Curcumin quantum dots prepared based on triaminoguanidine hydrochloride

[0095] A mixed solution of pure water and DMAC was used as a solvent (volume ratio of 1:1), and curcumin was added and stirred at 30°C for 30 minutes, followed by ultrasonication for 30 minutes to make it uniformly dissolved. Then, triaminoguanidine hydrochloride was added at a molar ratio of 2:3 to curcumin and refluxed at 180°C for 12 hours.

[0096] The excess solvent was removed by vacuum rotary evaporation, and the insoluble matter was removed by soaking in pure water, followed by freeze drying (vacuum, -40 degrees, freezing time 10 hours) to obtain the curcumin quantum dots (No. 4) of Example 4. The weight average molecular weight of the curcumin quantum dots measured by gel permeation chromatography (GPC) was 1511 Da, and GPC was measured at room temperature on a Tosoh High-Temperature EcoSEC detector using 1,2,4-trichlorobenzene as an eluent.

[0097] Comparative Example 1

[0098] A mixed solution of pure water and DMAC was used as the solvent (volume ratio 1:1), and curcumin was added, stirred at 30°C for 30 minutes, and then ultrasonicated for 30 minutes to make it uniformly dissolved. After refluxing at 180°C for 12 hours, DCC dehydrating agent was added and stirred for 5 hours.

[0099] After filtering out the dehydrating agent, the excess solvent was removed by vacuum rotary evaporation, and the insoluble matter was removed by soaking in pure water, followed by freeze drying (vacuum, -40 degrees, freezing time 12h) to obtain the curcumin sample of Comparative Example 1 (also referred to as pure curcumin).

[0100] Comparative Example 2

[0101] Use a mixed solution of pure water and DMAC as solvent (volume ratio 1:1), add curcumin, stir at 30°C for 30 minutes, and then ultrasonicate for 30 minutes to make it evenly dissolved. Then add ethylamine at a molar ratio of 1:1 to curcumin, reflux at 180°C for 12 hours, add DCC dehydrating agent and continue stirring for 5 hours.

[0102] After filtering out the dehydrating agent, the excess solvent was removed by vacuum rotary evaporation, and the insoluble matter was removed by soaking in pure water, followed by freeze drying (vacuum, -40 degrees, freezing time 12h) to obtain the curcumin sample of Comparative Example 2.

[0103] Test Case

[0104] 1. Determination of Fourier transform external light spectrum

[0105] The IR spectra of the curcumin quantum dots of Examples 1-4 were measured by the Fourier transform spectrometer.

[0106] 2. Determination of UV-Visible Absorption Spectrum

[0107] The UV-visible absorption spectra of the above Examples 1 to 4 were measured by the above UV-visible absorption spectrum. The spectra were measured in 5 mM sodium phosphate buffer solution (pH 7.4).

[0108] 3. Determination of transmission electron microscopy images

[0109] The transmission electron microscopic images of Examples 1-4 and Comparative Examples 1-2 were obtained by the transmission electron microscope described above.

[0110] 4. Determination of Fluorescence Images

[0111] A549 cells (human lung non-small cell carcinoma cells obtained from Nanjing Kebai Biotechnology Co., Ltd.) in the logarithmic growth phase were digested with trypsin to obtain a cell suspension, which was diluted to 5×10 4 / mL, 600 μl of cell suspension was added to a 6-well plate, 900 μl of complete culture medium was added after the cells adhered to the wall and incubated for 24 hours, the culture medium was discarded, 1 ml of 40 μM sample of Example 1-4 was added, PBS was washed after incubation for 24 hours, DAPI staining was performed and PBS was washed, and the cell morphology and fluorescence imaging were observed using a laser confocal microscope system at an excitation wavelength of 405 nm. The obtained fluorescence image is shown in FIG. Fig.11 shown.

[0112] 5. Cytotoxicity Assay

[0113] The CCK-8 kit was used to determine the toxicity of the samples of the reference example and Examples 1-4 to human lung non-small cell carcinoma A549 cells. 4 / ml A549 cell suspension was added to a 96-well plate at 100 μl per well, and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours. Different concentrations (1, 5, 10, 20, 40 μM) of the sample solutions of Comparative Example 1 (curcumin) and Examples 1-4 prepared with sterile deionized water were added to each well. After further incubation for 24 hours, CCK-8 reagent was added to determine the cell survival rate, and the IC50 value was calculated (expressed as the optical density OD value at 450 nm, the lower the OD value, the higher the tumor toxicity to the cells). The results are shown in Figure 2. Figure 7 shown.

[0114] 6. Cell Apoptosis Detection

[0115] 2.5×10 5 / well A549 cell suspension was inoculated on a 6-well plate, incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours, 0.5 ml of 40 μM samples of Comparative Example 1 and Examples 1-4 were added, incubated for 6 hours without serum, and then incubated for 48 hours with serum, the culture medium was discarded, and the cell suspension was prepared by trypsin digestion after washing with PBS, and then stained with 1 μL of dead and live dye and incubated on ice in the dark for 30 minutes, washed with PBS and resuspended, and Fc receptors were blocked, and after antibody incubation, the cells were washed with PBS and resuspended and then loaded on a flow cytometer. The results are shown in Figure 8 shown.

[0116] 7. Scratch test

[0117] A549 cells were seeded at 80% density in a 6-well plate overnight, and scratches of similar size were artificially made in the 6-well plate using a Tip. After washing with PBS, 0.5 ml of 40 μM samples of the reference example, embodiments 1-4, and comparative example 1 were added for treatment. After 24 hours, the scratch width was observed and measured, and the average scratch width was obtained by measuring three random areas, and the migration distance was calculated, and the migration rate of the cells was calculated. The results are shown in Fig. 9 shown.

[0118] 8. Transwell invasion assay

[0119] A549 cells were starved for 12 hours after serum withdrawal to prepare a cell suspension, and 1000 A549 cells were counted and treated with 200 μL of culture medium containing 40 μM of the samples of the reference example, Examples 1-4 and Comparative Example 1, respectively. The cells were added to the Transwell microporous filter culture chamber, and 600 μL of culture medium containing 15% FBS was added to the lower chamber of the 24-well plate. After incubation for 24 hours, the chamber was taken out, the culture medium was discarded, and DPBS was washed. The residual cells on the upper layer of the chamber were wiped off with a cotton swab, and the cells were fixed with ethanol, stained with crystal violet, and counted under a microscope. The results are shown in Fig.10 shown.

[0120] 9. Water solubility test

[0121] Take a certain amount of sample powder (about 10 mg) of Comparative Examples 1-2 and Examples 1-4, respectively, and add them to 10 ml of deionized water. Oscillate at a speed of 100 rpm for 2 hours in a constant temperature oscillator (37 ° C). Use high performance liquid chromatography (HPLC) to determine the concentration of curcumin in the solution. Quantitative analysis by internal standard method.

[0122] Discussion and Results

[0123] Table 1 shows the aqueous solution states and curcumin solubility data of the samples of Comparative Examples 1-2 and Examples 1-4. Figure 1 The solubility comparison diagram of curcumin (No. 0) of comparative example 1 and curcumin quantum dots prepared in examples 1-4 (corresponding to No. 1, No. 2, No. 3 and No. 4, respectively) in pure water is shown in Table 1 and Figure 1 As shown, compared with Comparative Examples 1 and 2, the water solubility of the curcumin quantum dots of Examples 1-4 is greatly improved and enhanced.

[0124] The curcumin quantum dots of Example 2 have the best solubility test results.

[0125] Table 1

[0126] sample Solution state Solubility (mg / 100ml water) Comparative Example 1 Turbid yellow dispersion About 0.1 Comparative Example 2 Turbid yellow dispersion About 0.5 Example 1 Orange-red transparent solution About 20.3 Example 2 Orange-yellow transparent solution About 11.7 Example 3 Orange-yellow transparent solution About 15.8 Example 4 Orange-yellow transparent solution About 10.2

[0127] The microscopic morphology and size of the curcumin quantum dots prepared in Examples 1-4 are as follows: Figure 2 A, 2B, 2C and 2D. Figure 2 It can be seen from A, 2B, 2C and 2D that the curcumin quantum dots prepared in Examples 1-4 are uniform, dispersed single spheres with an average diameter of 2nm-5nm. Curcumin quantum dots cannot be obtained in Comparative Examples 1 and 2. Specifically, the powder particles obtained in Comparative Example 1 have an average diameter of 200-400 μm after drying; the powder particles obtained in Comparative Example 2 have an average diameter of 500nm-700nm after drying.

[0128] like Figure 3 As shown, through the FTIR infrared spectrum analysis on the left, it can be observed that the characteristic peak belonging to the -C=N- group of Example 1, combined with the weakening of the carbonyl peak of curcumin, proves the introduction of multi-amino ligands and the formation of Schiff bases; Figure 3 The change analysis of the UV-mid-UV absorption on the right side of the middle can also prove the introduction of multi-amino complexes and the formation of quantum dots. Figure 4 As shown, through the FTIR infrared spectrum analysis on the left, it can be observed that the characteristic peak of the sample of Example 2 belongs to the -C=N- group, combined with the weakening of the carbonyl peak of curcumin, it is proved that the introduction of multi-amino ligands and the formation of Schiff bases; Figure 4The change of UV absorption in the right side of the UV spectrum can also be analyzed to see the introduction of multi-amino complexes and the formation of quantum dots. Figure 5 As shown, through the FTIR infrared spectrum analysis on the left, it can be observed that the characteristic peak of the sample of Example 3 belongs to the -C=N- group, combined with the weakening of the carbonyl peak of curcumin, it is proved that the introduction of multi-amino ligands and the formation of Schiff bases; Figure 5 The change of UV absorption in the right side of the UV spectrum can also be analyzed to see the introduction of multi-amino complexes and the formation of quantum dots. Figure 6 As shown, through the FTIR infrared spectrum on the left, the characteristic peak of the -C=N- group belonging to the sample of Example 4 can be observed. Combined with the weakening of the carbonyl peak of curcumin, the introduction of multi-amino ligands and the formation of Schiff bases are proved; by analyzing the changes in ultraviolet absorption in ultraviolet, the introduction of multi-amino complexes and the formation of quantum dots can also be confirmed.

[0129] Then, the samples of Comparative Example 1 and Examples 1-4 were used to detect cytotoxicity and cell apoptosis according to the above method. The test results were as follows: Figure 7 and Figure 8 As shown. Figure 7 It can be seen that with the increase of sample concentration, especially when the dose is 10 μM or higher, the curcumin quantum dots of Example 1 have significantly higher tumor cell toxicity, and it is expected to have better anti-tumor activity in vivo. Figure 8 It was demonstrated that compared with Comparative Example 1, at each concentration, the curcumin quantum dots of Examples 1-4 all showed significantly better tumor cell apoptosis effects, including late apoptosis effects and early apoptosis effects. In particular, Example 1 showed better effects in both late apoptosis and early apoptosis. Example 2 showed more normal cells and cell fragments. Examples 3 and 4 only had more obvious effects in late apoptosis of tumor cells.

[0130] The samples of Comparative Example 1 and Examples 1-4 were tested for their anti-tumor cell migration and invasion using scratch test and Transwell test. The results were as follows: Fig. 9 and Fig.10 As shown. Fig. 9 As shown, compared with Comparative Example 1, the turmeric quantum dots of Examples 1-4 showed significantly better anti-tumor cell migration ability, and this anti-migration ability increased with the increase of sample concentration. In particular, the curcumin quantum dots of Example 1 showed the best anti-tumor cell migration ability at various sample concentrations, and even substantially prevented tumor cell migration at a concentration of 80 μM.

[0131] like Fig.10As shown, compared with Comparative Example 1, the turmeric quantum dots of Examples 1-4 showed significantly better anti-tumor cell invasion ability, and this anti-invasion ability also increased with the increase of sample concentration. Similarly, the curcumin quantum dots of Example 1 had the best anti-tumor cell invasion ability.

[0132] The staining efficacy of the samples of Examples 1-4 on tumor cells is as follows: Fig.11 As shown. Fig.11 As shown, the curcumin quantum dots of Examples 1-4 of the present invention can display excellent fluorescent images of tumor cells, and can diagnose and identify tumor cells, thereby realizing integrated diagnosis and treatment.

[0133] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the embodiments of the present invention, rather than to limit them. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the above embodiments, or replace some of the technical features therein by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention. Any changes or replacements that can be easily thought of by those skilled in the art within the technical scope disclosed in the present invention should be covered within the protection scope of the present invention.

Claims

1. A curcumin quantum dot, characterized in that: The invention relates to a reaction product formed by a Schiff base reaction between curcumin and a polyamino ligand in the presence of a dehydrating agent, wherein the average diameter of the curcumin quantum dots is 1-30 nm.

2. The curcumin quantum dots according to claim 1, characterized in that: The number of amino groups in the polyamino ligand is equal to or greater than 2.

3. The curcumin quantum dots according to claim 1, characterized in that: The polyamino ligand is selected from one of ethylenediamine, p-phenylenediamine, o-phenylenediamine, benzene-1,2,3-triamine, triaminoguanidine hydrochloride and 2,4,6-triaminopyrimidine or any combination thereof.

4. The curcumin quantum dots according to any one of claims 1 to 3, characterized in that: The dehydrating agent is selected from one or more of DCC and concentrated sulfuric acid.

5. The curcumin quantum dots according to any one of claims 1 to 3, characterized in that The reaction product is a Schiff base polymer formed by the two carbonyl groups of curcumin and the multiple amino groups of the polyamino ligand through a Schiff base reaction, wherein the weight average molecular weight of the Schiff base polymer is 900-2000 Da.

6. A method for preparing curcumin quantum dots according to claim 1, characterized in that: The following steps are involved: S1-providing a solution of curcumin in a reaction solvent; S2-reacting curcumin with polyamino ligands in the solution for a period of time; Then S3-adding a dehydrating agent to continue the reaction, thereby obtaining a solution of the reaction product; and S4-The reaction product is purified and dried to obtain curcumin quantum dots.

7. The preparation method according to claim 6, characterized in that: The reaction solvent is selected from one or more of water, ethanol, N,N-dimethylacetamide, N,N-dimethylformamide and dimethyl sulfoxide.

8. The preparation method according to claim 6 or 7, characterized in that: The reaction temperature of step S2 is 25-210° C., the reaction time is 10-20 hours, and the reaction process includes hydrothermal reaction or reflux reaction; The reaction temperature of step S3 is 25-210° C., and the reaction time is 1-10 hours; The purification in step S4 includes filtering to remove the dehydrating agent, removing excess solvent and washing with water; as well as The drying of step S4 includes freeze drying or heat drying, the temperature of freeze drying is -200 to -4°C, and the temperature of heat drying is 25 to 100°C.

9. The preparation method according to claim 6, characterized in that The weight ratio of curcumin to amino ligand is (10-90):(10-50).

10. Use of the curcumin quantum dots according to any one of claims 1 to 5 in the preparation of an integrated diagnosis and treatment reagent for tumors, wherein the integrated diagnosis and treatment comprises staining and marking of tumor cells and therapeutic inhibition of tumor cells.

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