CN104826127A - Preparation method and applications of photo-thermal and photodynamic co-used antitumor drug delivery system taking gold nano star mediated by folic acid as carrier - Google Patents

Abstract

The present invention relates to the preparation method and application of a photothermal and photodynamic combined anti-tumor drug delivery system using folic acid-mediated gold nanostars as carriers. It can effectively solve the problem of existing anti-tumor drugs with large toxic side effects, poor biocompatibility, and poor targeting. To solve the problem of poor stability, the method is to connect the reactive amino and carboxyl groups on the surface of gold nanostars through covalent bonds, and then connect the targeting molecules through amide reaction to obtain an actively targeted gold nanostar solution; then through the targeting The amino group at the end of the compound undergoes a chemical reaction with a near-infrared fluorescent probe to obtain a gold nanostar solution with folic acid targeting and near-infrared fluorescence imaging. The anti-tumor drug doxorubicin is then directly loaded on the surface of the gold nanostar through electrostatic adsorption. That is, the body of the present invention has good physical and chemical stability, simple preparation process, abundant sources of raw materials, low cost, few toxic and side effects, and can effectively inhibit the proliferation of tumor cells.

Description

Preparation method and application of folic acid-mediated gold nanostar-carrier drug delivery system combined with photothermal and photokinetic anti-tumor

technical field

The invention relates to medicine, in particular to a preparation method and application of a drug delivery system using folic acid-mediated gold nanostar as a carrier for combined photothermal and photokinetic anti-tumor.

Background technique

Photo-thermal therapy (Photo-thermal Therapy, PTT) is to change the environment of tumor cells by heating, so that tumor cells can degenerate and necrosis, so as to achieve the purpose of treatment. For example, various gold nanostructures, including gold nanorods , nanoshells, nanocages, hollow nanospheres, especially branched gold nanostars, have been proven to have surface plasmon resonance properties in the near-infrared region, and thus generate excessive heat through near-infrared laser irradiation, thereby killing tumor cells.

Photodynamic therapy (Photodynamic Therapy, PDT) is the use of tumor cells to selectively absorb photosensitizers, and then use non-thermal lasers of specific wavelengths to irradiate the lesion, so that the photosensitizers in tumor tissues undergo intense photochemical reactions, thereby selectively destroying tumors cell. It is a non-invasive or minimally invasive, non-thermal treatment method that uses photochemical reactions to cause damage to target tissues and cells. As an excellent photosensitizer, gold nanostars can generate active oxygen under near-infrared light irradiation, including peroxides, such as H ₂ O ₂ , singlet oxygen (Singlet Oxygen), epoxide, etc., And it is highly reactive to produce active oxygen under light, which can show obvious cytotoxicity, such as breaking DNA in cells, inhibiting cell mitosis and growth, and inhibiting the activity of cellular proteolytic enzymes. It can be inferred that gold nanostars can be used for combined photothermal and photodynamic therapy of deep tumor tissue.

Because of the advantages of easy mass synthesis and large specific surface area, gold nanostars can increase the content of entrapped drugs. At the same time, because biological systems are highly transparent to near-infrared light in the range of 700-1100nm, gold nanostars are within this range. It has high absorption characteristics, so it can use its photothermal conversion characteristics within this range to perform near-infrared laser hyperthermia on tumors. It is possible to form a multifunctional drug delivery system that integrates chemotherapy, near-infrared hyperthermia and photodynamic therapy by chemically modifying gold nano-star-loaded anti-tumor drugs.

Because gold nanostars are prone to aggregation and agglomeration in the presence of high salt and certain biomolecules (nucleic acid, protein, etc.), the stability is poor. In order to improve the stability and biocompatibility of gold nanostars, mercapto-polyethylene glycol and polyetherimide were linked to the surface of gold nanostars through the sulfur-gold bond formed by thiol and gold. Linking water-soluble polyethylene glycol with long circulation characteristics to the surface of gold nanostars can not only take advantage of the steric hindrance effect, improve the stability of gold nanostars, prolong the circulation time in vivo, and reduce toxicity, but also the polyethylene glycol carried Different side chain structures provide effective modification sites for the targeted modification of the surface of gold nanostars. In addition, mercapto-polyetherimide contains more protonated nitrogen atoms of multi-level ammonia, which can capture a large number of protons in acidic lysosomes, resulting in osmotic swelling and rupture of lysosomes, and then the endocytosed drugs Rapidly released into the cytoplasm to achieve lysosomal escape.

Molecular targeted therapy is a new method of tumor treatment, which can specifically act on target molecules that play a key role in the development of tumors, or use receptors highly expressed in tumors to deliver targeted drugs through ligands to tumor cells, so as to achieve the purpose of treating tumors and reducing systemic side effects. The folate receptor is a high-affinity receptor that can mediate endocytosis and take folic acid into the cytoplasm of eukaryotic cells. It has entered clinical application research as a molecular target for tumor therapy. The body has a high affinity for the folic acid receptor overexpressed on the surface of malignant tumor cells, but it is almost not expressed in most normal tissues. At the same time, the modified drug carrier is a special targeted drug delivery system that can Actively deliver drugs to specific sites.

In the study of tumor drugs, the use of fluorescent probes to label drugs can directly observe whether the drug targets the tumor, the optimal targeting time, and the accumulation of the drug in other organs and tissues of the animal at the living level. Time for organizational analysis. Fluorescent probes such as indole-5.5-cyanine-N-hydroxysuccinimide, indocyanine green, and phthalocyanine can not only be used for in vivo imaging but also absorb near-infrared light. The electrons in the molecule transition from the ground state to the excited state. The molecules in the excited state then release heat, which has a hyperthermic effect. Most of the fluorescent probes commonly used in in vivo imaging are traditional organic dye molecules, such as indocyanine green, phthalocyanine, methylene blue, rhodamine, fluorescein isothiocyanate, and these organic dye molecules have their own Some disadvantages, such as poor photostability, low sensitivity and poor targeting, thus limit its further applications. The above-mentioned defects can be overcome with the help of targeted nanoparticle carriers, which has obvious advantages in the diagnosis and targeted therapy of tumors.

Doxorubicin is a broad-spectrum chemotherapeutic drug, which is commonly used clinically to treat malignant tumors such as breast cancer, children's solid tumors, soft tissue tumors, and nauseated lymphoma. Clinical studies in recent years have found that doxorubicin has been resistant to tumor cells in clinical applications, and its cytotoxicity to normal tissues limits its further application. The most common toxicity of long-term use of these drugs is damage to the myocardium, which can induce congestive heart failure. In order to overcome the above shortcomings, adriamycin can be directly loaded on the surface of gold nanostars through electrostatic adsorption (Au-N) to construct a drug delivery system targeting anti-tumor with gold nanostars as carriers.

The currently constructed anti-tumor drug delivery system using gold nanostars as carriers has not been reported as a heat-sensitizer and photosensitizer in the combination therapy of tumors. It has the advantages of rapid curative effect, small side effects, and simple method. At the same time, using gold nanostars as carriers to load near-infrared fluorescent dyes can improve the stability of fluorescent dyes, prolong the imaging time in vivo, and endow them with targeting through the carrier system. Compared with the dye direct imaging method, the application range is wider, and the current application is mainly concentrated in the stage of animal experiments in the laboratory. Therefore, it is a technical problem that the industry hopes to solve to develop a drug delivery system that uses folic acid-mediated gold nanostars as a carrier for photothermal and photokinetic combined anti-tumor with statistical significance and practical clinical significance.

Contents of the invention

In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a preparation method and application of a drug delivery system with folic acid-mediated gold nanostars as the carrier of photothermal and photodynamic combined anti-tumor, which can effectively The invention solves the problems of large toxic and side effects, poor biocompatibility and poor targeting of existing antitumor drugs.

The technical solution solved by the present invention is to connect the reactive amino group and carboxyl group on the surface of the gold nanostar through a covalent bond, and then connect the targeting molecule through an amide reaction to obtain an actively targeted gold nanostar solution; Carry out a chemical reaction to the amino group at the end of the compound and a near-infrared fluorescent probe to obtain a gold nanostar solution with folic acid targeting and near-infrared fluorescence imaging; then directly load the anti-tumor drug doxorubicin on the surface of the gold nanostar through electrostatic adsorption , that is to obtain a folic acid-mediated multifunctional gold nanostar drug delivery system for combined photothermal and photokinetic anti-tumor. The mass ratio of doxorubicin to gold nanostar is 1.5-3:1. The specific preparation method is as follows:

(1) Preparation of aminated gold nanostars: Add 10-20 mL of a gold nanostar solution with a concentration of 50 μg/mL at 18-25°C and a magnetic stirring condition of 300-720 r/min, and then add a concentration of 0.1- 1-5 mL of 10.0 mg/mL amino compound was stirred in the dark for 6-12 h, then centrifuged at 10,000 rpm for 30 min at high speed, and then reconstituted with ultrapure water to obtain an aminated gold nanostar solution;

The amino compound is one or a mixture of NH ₂ -PEG2000-SH and PEI-SH;

(2) Preparation of targeted gold nanostars: Weigh 15-20 mg of folic acid, 10-15 mg of N, N'-dicyclohexylcarbimide, and 7.5-10.5 mg of N-hydroxysuccinimide, and then Add 2.5-5.0 mL of the reaction solution in turn, mix and dissolve, and then activate the carboxyl group, that is, at 40-50 ° C, 300-720 r/min, avoid light and magnetically stir the reaction for 8-24 hours, and then centrifuge the obtained solution at 10,000 rpm for 30 minutes The precipitate was obtained, and the precipitate was redissolved with ultrapure water, added to the aminated gold nanostar solution prepared in step (1) for acylation reaction, stirred and reacted at room temperature in the dark for 12-24 hours, and then centrifuged at 10000 rpm for 30 minutes at high speed to remove free small Molecules, to obtain folic acid-targeted gold nanostar solution containing amino groups at the end;

The reaction solution is one of dimethyl sulfoxide, phosphate buffer (neutral) and acetone;

(3) Preparation of gold nanostar solution with near-infrared fluorescence imaging and targeting: Dissolve 0.01-2 mg fluorescent probe in 0.01-1 mL of reaction solvent, add folic acid-targeted gold nanostars prepared in step (2) Carry out amide reaction in the star solution, stir and react at room temperature in the dark for 12-24 hours, then ultracentrifuge at 10,000rpm for 30 minutes to obtain a precipitate, and redissolve the precipitate in 0.01-1mL of ultrapure water to obtain gold nanostars with targeting properties and near-infrared fluorescence. Imaging anti-tumor drug delivery systems;

The fluorescent probes are indole-5-cyanine (abbreviated as cy5), indole-5.5-cyanine (abbreviated as cy5.5) and indole-5.5-cyanine-N-hydroxysuccinimide (abbreviated as cy5.5-NHS);

Described reaction solvent is a kind of in dimethyl sulfoxide, phosphate buffer saline and acetone;

(4) Preparation of doxorubicin-loaded gold nanostar solution: the targeted gold nanostar solution with near-infrared fluorescence imaging prepared in step (3) was dried to obtain a powder, and the powder and doxorubicin were mixed at a mass ratio of 1:1.5 Add ～3.0 to 10～40 mL of reaction solvent in turn, under the protection of nitrogen, ultrasonically dissolve, and stir at 18～25°C at 300-720r/min in the dark for 24～48h to obtain a mixed solution, and then mix the obtained The solution was ultracentrifuged at 10,000rpm for 30min (to remove unreacted doxorubicin and dimethyl sulfoxide) to obtain a precipitate, which was redissolved in ultrapure water and dried to obtain the folic acid-mediated gold Nanostar-based drug delivery system;

The drying is one of freeze drying, vacuum drying and drying.

The application of the photothermal and photodynamic combined anti-tumor drug delivery system mediated by folic acid-mediated gold nanostars in the preparation of active targeting anti-tumor drug delivery carriers, or in the preparation of anti-tumor photodynamic or photothermal therapy The application of thermosensitizers, or in the preparation of near-infrared laser irradiation, the application of triggering the targeted and rapid release of chemical drugs in tumor tissue drugs, or the preparation of drug delivery systems for combined photothermal and photokinetic anti-tumor in the preparation of biological In vivo intravital fluorescence imaging for pharmaceutical applications.

The folic acid-mediated multifunctional gold nanostar of the present invention is used in a photothermal and photokinetic combined anti-tumor drug delivery system, and can be loaded with chemotherapy drugs. Gold nanostars have photothermal conversion characteristics and the ability to generate active oxygen. Combining laser irradiation with tumor sites, using gold nanostars as thermosensitizers, heat generation under laser irradiation can promote the targeted release of drugs at tumor sites, thereby achieving Combined anti-tumor effects of hyperthermia and chemotherapy; the body of the present invention has good physical and chemical stability, simple preparation process, rich source of raw materials, low cost, less toxic and side effects, can effectively inhibit tumor cell proliferation, and is a drug carrier in anti-tumor targeted therapy on innovation.

Detailed ways

The specific implementation of the present invention will be described in detail below in conjunction with the examples.

The present invention can be provided by the following examples in concrete implementation.

Example 1

In concrete implementation, the present invention can be realized by the following steps:

(1) Preparation of aminated gold nanostar solution: Add 20 mL of gold nanostar solution with a concentration of 50 μg/mL, and then add NH ₂ -PEG2000 with a concentration of 1 mg/mL under the condition of 25°C and 540 r/min magnetic stirring -SH solution 1mL, stirred at room temperature and protected from light for 12h, then centrifuged at 10000rpm for 30min at high speed to obtain a precipitate, which was redissolved in 21mL of ultrapure water to obtain an aminated gold nanostar (GNSTs-PEG) solution;

(2) Preparation of targeted gold nanostars: Weigh 19.91 mg of folic acid, 13.94 mg of N,N'-dicyclohexylcarbimide, and 10.36 mg of N-hydroxysuccinimide (weight ratio: folic acid : N,N'-dicyclohexylcarboimide: N-hydroxysuccinimide=1︰0.7︰0.52), then add to 4mL dimethyl sulfoxide in turn, mix and dissolve, and then activate the carboxyl group, that is React at 50°C and 480r/min with magnetic stirring for 12 hours in the dark, then centrifuge the resulting solution at 10,000 rpm for 30 minutes to obtain a precipitate, redissolve the precipitate in ultrapure water, and add it to the aminated gold nanostar solution prepared in step (1) for acylation The reaction was carried out at room temperature in the dark and stirred for 24 hours, then centrifuged at 10,000 rpm for 30 minutes at high speed to remove free small molecules and obtain a folic acid-targeted gold nanostar (GNSTs-PEG-FA) solution containing amino groups at the end;

(3) Preparation of gold nanostar solution with near-infrared fluorescence imaging and targeting: Dissolve 1 mg of fluorescent probe indole-5.5-cyanine-N-hydroxysuccinimide (cy5.5-HNS) in 1 mL of di In methyl sulfoxide, add the folic acid targeting gold nanostar solution prepared in step (2) for amide reaction, stir and react at room temperature in the dark for 24 hours, then ultracentrifuge at 10,000rpm for 30 minutes to obtain a precipitate, and redissolve the precipitate with 0.5mL of ultrapure water. A gold nanostar (GNS-PEG-FA-cy5.5) solution with near-infrared fluorescence imaging and targeting using gold nanostar as a carrier;

(4) Preparation of gold nanostar solution loaded with doxorubicin: the gold nanostar solution with near-infrared fluorescence imaging and targeting prepared in step (3) was freeze-dried to obtain a powder, and the powder and doxorubicin were mixed in a mass ratio Add 1:2 to 24mL dimethyl sulfoxide sequentially, under the protection of nitrogen, ultrasonically dissolve, and stir at 25°C at 480r/min in the dark for 24h to obtain a mixed solution, and then ultracentrifuge the obtained mixed solution at 10000rpm After 30 minutes (to remove unreacted doxorubicin and dimethyl sulfoxide), the precipitate was obtained. The precipitate was redissolved in ultrapure water, and freeze-dried to obtain folic acid-mediated gold nanostars combined with photothermal and photokinetic antitumor. Carrier drug delivery system (GNSTs/DOX-PEG-FA-cy5.5) powder.

Example 2

In concrete implementation, the present invention can be realized by the following steps:

(1) Preparation of aminated gold nanostars: Add 15 mL of gold nanostar solution with a concentration of 50 μg/mL, and then add 3 mL of PEI-SH with a concentration of 5 mg/mL at 20 °C and 600 r/min magnetic stirring conditions, Stir the reaction in the dark for 8 hours, then centrifuge at a high speed of 10000 rpm for 30 minutes, and then redissolve with ultrapure water to obtain an aminated gold nanostar solution;

(2) Preparation of targeted gold nanostars: Weigh 18 mg of folic acid, 12 mg of N, N'-dicyclohexylcarbimide, and 9 mg of N-hydroxysuccinimide, and then add 4 mL of phosphate buffer in sequence In, mix and dissolve, and then activate the carboxyl group, that is, at 45°C, 500r/min, shielded from light and magnetically stirred for 16h, then centrifuge the resulting solution at 10,000 rpm for 30min to obtain a precipitate, redissolve the precipitate with ultrapure water, and add the step ( 1) Acylation reaction was carried out in the prepared aminated gold nanostar solution, stirred at room temperature in the dark for 16 hours, then centrifuged at 10,000 rpm for 30 minutes to remove free small molecules, and a folic acid-targeted gold nanostar solution containing amino groups at the end was obtained;

(3) Preparation of gold nanostar solution with near-infrared fluorescence imaging and targeting: Dissolve 1.5 mg of fluorescent probe indole-5-cyanine in 0.8 mL of phosphate buffer, add folic acid prepared in step (2) Carry out amide reaction in the targeting gold nanostar solution, stir and react at room temperature in the dark for 16 hours, then ultracentrifuge at 10,000rpm for 30min to obtain a precipitate, and redissolve the precipitate in 0.8mL of ultrapure water to obtain a gold nanostar with targeting and near-infrared properties. Fluorescence imaging anti-tumor drug delivery system;

(4) Preparation of doxorubicin-loaded gold nanostar solution: The targeted gold nanostar solution with near-infrared fluorescence imaging prepared in step (3) was vacuum-dried to obtain a powder, and the powder and doxorubicin were mixed in a mass ratio of 1: 2.5 Add it to 35mL of phosphate buffer in turn, dissolve it under nitrogen protection, ultrasonically dissolve, stir and react at 600r/min in the dark for 30h at 20°C to obtain a mixed solution, and then ultracentrifuge the obtained mixed solution at 10000rpm for 30min (remove unreacted doxorubicin and phosphate buffer) to obtain a precipitate, which was redissolved in ultrapure water and dried in vacuum to obtain a drug delivery system with folic acid-mediated gold nanostars as a carrier for combined photothermal and photokinetic anti-tumor powder.

Example 3

In specific implementation, the present invention can also be realized by the following steps:

(1) Preparation of aminated gold nanostars: Add 15 mL of gold nanostar solution with a concentration of 50 μg/mL, and then add 3 mL of amino compounds with a concentration of 5.0 mg/mL under the condition of 21 ° C and 510 r/min magnetic stirring, Stir the reaction in the dark for 9 hours, then centrifuge at a high speed of 10000 rpm for 30 minutes, and then redissolve with ultrapure water to obtain an aminated gold nanostar solution;

(2) Preparation of targeted gold nanostars: Weigh 17.5 mg of folic acid, 12.5 mg of N, N'-dicyclohexyl carboximide, and 9 mg of N-hydroxysuccinimide, and then add 3.5 mL of them in turn to react In the solution, mix and dissolve, and then activate the carboxyl group, that is, at 45°C and 510r/min to avoid light and magnetically stir for 16h, then centrifuge the resulting solution at 10,000 rpm for 30min to obtain a precipitate, which is redissolved with ultrapure water, and added to the step (1) Acylation reaction was carried out in the prepared aminated gold nanostar solution, stirred at room temperature in the dark for 18 hours, then centrifuged at 10,000 rpm for 30 minutes to remove free small molecules, and a folic acid-targeted gold nanostar solution containing amino groups at the end was obtained;

(3) Preparation of gold nanostar solution with near-infrared fluorescence imaging and targeting: Dissolve 1 mg of fluorescent probe in 0.5 mL of reaction solvent, add folic acid targeting gold nanostar solution prepared in step (2) for amide Reaction, stirring at room temperature and avoiding light for 18 hours, and then ultracentrifuging at 10,000 rpm for 30 minutes to obtain a precipitate, which was redissolved in 0.5 mL of ultrapure water to obtain an anti-tumor drug delivery system using gold nanostars as a carrier with targeting and near-infrared fluorescence imaging;

(4) Preparation of doxorubicin-loaded gold nanostar solution: the targeted gold nanostar solution with near-infrared fluorescence imaging prepared in step (3) was dried to obtain a powder, and the powder and doxorubicin were mixed at a mass ratio of 1:1.7 Sequentially added to 25mL of reaction solvent, under the protection of nitrogen, ultrasonically dissolved, stirred and reacted at 510r/min in the dark for 36h at 21°C to obtain a mixed solution, and then ultracentrifuged the obtained mixed solution at 10000rpm for 30min (to remove unreacted Doxorubicin and reaction solvent) to obtain a precipitate, redissolve the precipitate with ultrapure water, and dry to obtain a drug delivery system powder with folic acid-mediated gold nanostar as the carrier for combined photothermal and photodynamic anti-tumor.

Example 4

In specific implementation, the present invention can also be realized by the following steps:

(1) Preparation of aminated gold nanostars: Add 13-17mL of gold nanostar solution with a concentration of 50μg/mL at 20-22°C and magnetic stirring at 500-600r/min, and then add 6-8mg /mL amino compound 2-3mL, stirred and reacted in the dark for 8-10 h, then centrifuged at 10,000 rpm for 30 min, and then re-dissolved with ultrapure water to obtain an aminated gold nanostar solution;

(2) Preparation of targeted gold nanostars: Weigh 17-18 mg of folic acid, 12-13 mg of N, N'-dicyclohexylcarbimide, and 8-9.5 mg of N-hydroxysuccinimide, and then Add 3-4mL reaction solution in turn, mix and dissolve, and then activate the carboxyl group, that is, at 43-47°C, 500-600r/min, avoid light and magnetically stir for 12-20h, and then centrifuge the resulting solution at 10,000 rpm for 30min to obtain Precipitate, redissolve the precipitate with ultrapure water, add the aminated gold nanostar solution prepared in step (1) for acylation reaction, stir at room temperature in the dark for 16-20h, then centrifuge at 10,000 rpm for 30min at high speed to remove free small molecules , to obtain a folic acid-targeted gold nanostar solution containing an amino group at the end;

(3) Preparation of gold nanostar solution with near-infrared fluorescence imaging and targeting: Dissolve 0.8-1.2 mg of fluorescent probe in 0.4-0.6 mL of reaction solvent, add folic acid-targeted gold nanoparticles prepared in step (2) Carry out amide reaction in the star solution, stir the reaction for 16-20 hours at room temperature in the dark, and then ultracentrifuge at 10,000 rpm for 30 minutes to obtain a precipitate. Fluorescence imaging anti-tumor drug delivery system;

(4) Preparation of doxorubicin-loaded gold nanostar solution: the targeted gold nanostar solution with near-infrared fluorescence imaging prepared in step (3) was dried to obtain a powder, and the powder and doxorubicin were mixed at a mass ratio of 1:1.8 -2.2 Add to 20-30mL reaction solvent in turn, under the protection of nitrogen, ultrasonically dissolve, and stir at 500-600r/min in the dark for 30-40h at 20-22°C to obtain a mixed solution, and then mix the obtained mixed solution Ultracentrifuge at 10,000rpm for 30min (remove unreacted doxorubicin and dimethyl sulfoxide) to obtain a precipitate, redissolve the precipitate in ultrapure water, and dry to obtain the folic acid-mediated gold nanometer combined with photothermal and photodynamic anti-tumor Star as carrier drug delivery system powder.

The particle size of the photothermal and photodynamic anti-tumor drug delivery system mediated by folic acid-mediated gold nanostars prepared by the present invention is 80-150nm, which is effectively used as a drug delivery carrier for active targeting anti-tumor; as a thermosensitizer In anti-tumor photodynamic or photothermal therapy; under near-infrared laser irradiation, the application of triggering the targeted and rapid release of chemical drugs to tumor tissue; in vivo fluorescence imaging. And achieved very satisfactory beneficial technical effects through tests, the relevant test data are as follows:

The folic acid-mediated multifunctional gold nanostar of the present invention is used in a photothermal and photokinetic combined anti-tumor drug delivery system, and its application in anti-tumor therapy is divided into two parts: in vitro and in vivo:

(1) In vitro: the multifunctional gold nanostar mediated by folic acid of the present invention is used in the photothermal and photokinetic combined anti-tumor drug delivery system to be added to the tumor cells for cultivation, and after 4 to 6 hours after administration, use a wavelength of 700 to 1100nm A broad wavelength light source or 808nm laser light was used for 1-5 minutes of light irradiation, and fresh medium was replaced for 24 hours to determine the survival rate of tumor cells.

The above-mentioned cancer cells are: various solid tumors that appear on the surface or inside of organs, lung cancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, leukemia, pancreatic cancer, cervical cancer, Laryngeal cancer, thyroid cancer, tongue cancer, brain tumor (intracranial tumor), small intestine tumor, gallbladder cancer, bile duct cancer, kidney cancer, prostate cancer, penile cancer, Cuiwan tumor, endometrial cancer, choriocarcinoma, vaginal malignancy Tumor, malignancy of the vulva, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, malignant melanoma.

(2) In vivo: The multifunctional gold nanostar mediated by folic acid of the present invention is used for photothermal and photokinetic combined anti-tumor drug delivery system intravenously into tumor-bearing mice, and 4-6 hours after administration, use 700-1100nm Wide-wavelength light source in the wavelength range or 808nm laser light for 1 to 5 minutes. After irradiation, use an infrared thermal camera to record the temperature of the irradiated part.

The tumor-bearing mice mentioned above are: various solid tumors appearing on the surface or inside of organs, lung cancer, nasopharyngeal cancer, esophageal cancer, gastric cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, leukemia, pancreatic cancer, cervical cancer Cancer, laryngeal cancer, thyroid cancer, tongue cancer, brain tumor (intracranial tumor), small intestine tumor, gallbladder cancer, bile duct cancer, kidney cancer, prostate cancer, penile cancer, Tsui Wan tumor, endometrial cancer, choriocarcinoma, One of vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, and malignant melanoma.

The novel folic acid-mediated multifunctional gold nanostar of the present invention is used in a photothermal and photokinetic combined anti-tumor drug delivery system. Patches, gels, implants, etc. Various preparation additives can be added to the preparation of the present invention, such as physiological saline, glucose, buffer solution and preservatives and the like. The administration method can be intravenous injection, intramuscular injection, intratumoral injection and subcutaneous injection, transdermal administration, internal implantation and the like.

The relevant test data are as follows:

1. Determination of the drug loading amount of the folic acid-mediated multifunctional gold nanostar used in the photothermal and photokinetic combined anti-tumor drug delivery system of the present invention:

The folic acid-mediated multifunctional gold nanostar of the present invention is used in a photothermal and photokinetic combined anti-tumor drug delivery system, and its drug loading is determined to be 1.76 mg/mL by ultraviolet spectrophotometry, indicating that the folic acid-mediated Multifunctional gold nanostars used in photothermal and photokinetic combined anti-tumor drug delivery system can be used as carriers of anti-tumor drugs.

2. Determination of the particle size and surface charge of the folic acid-mediated multifunctional gold nanostar of the present invention used in a photothermal and photokinetic combined anti-tumor drug delivery system:

In the present invention, the folic acid-mediated multifunctional gold nanostar is used for the determination of the particle size and surface charge of the photothermal and photokinetic combined anti-tumor drug delivery system, using a Nano-ZS90 laser particle size analyzer for measurement, and the refractive index is set is 1.590, the absorption coefficient is set to 0.010, the temperature is set to 25°C, the measurement mode is set to automatic, and the Z average statistical value is used as the measurement result. Three copies of each horizontal condensate were prepared, each was measured once, and the average value of the three measurements was taken as the measurement result. The dielectric constant is set to 79, the viscosity coefficient is set to 0.8872, the temperature is set to 25°C, and the measurement mode is set to automatic. Three copies of each horizontal condensate were prepared, each was measured once, and the average value of the three measurements was taken as the measurement result. The measured result is that the particle size is 80-150 nm, and the potential is -20-30mV.

3. In vitro anti-tumor activity of folic acid-mediated multifunctional gold nanostars used in photothermal and photokinetic combined anti-tumor drug delivery system:

In the present invention, the in vitro anti-tumor activity of folic acid-mediated multifunctional gold nanostars used in the drug delivery system of photothermal and photokinetic combined anti-tumor, MCF-7 breast cancer cells (provided by Shanghai Cell Bank) were used as the target to be investigated cancer cell. Human breast cancer cell MCF-7 was cultured in RPMI1640 medium containing 10% fetal bovine serum and 1% double antibody, and incubated in a cell culture incubator containing 5% CO2 at 37 °C, and the medium was changed every other day for 3 Digested and passaged with 0.25% trypsin (containing 0.02% EDTA) once every 4 days, and carried out related experiments when the cells grew to 70%-80%. Cells in logarithmic growth phase were used in all experiments. The toxic effect of folic acid-mediated multifunctional gold nanostars used in photothermal and photokinetic combined anti-tumor drug delivery system on MCF-7 cells was detected by SRB method. Take the cells in the logarithmic growth phase, routinely digest them into a single-cell suspension and count them, adjust the density of the cells to be tested to 5×10 ³ cells/well (the edge wells are filled with sterile PBS), spread 96-well plates, and add 200 μl to each well culture medium, and cultured in a cell incubator at 37°C with 5% CO2. After all the cells adhered to the wall for 24 h, the original medium was discarded, and the folic acid-targeted gold nanostar carrier loaded with folic acid-targeted gold nanostar carrier diluted in medium with a concentration gradient of (0.5, 1, 5, 10, 50, 100 μg/mL) was added to the experimental group. Doxorubicin drug system, set 3 to 5 duplicate wells for each concentration, add blank medium to the blank cell group, set 3 to 5 duplicate wells as the control group, continue to culture in the incubator for 24 h, and terminate the culture. Gently add 25 μl of pre-cooled 50% TCA to the surface of the culture medium for fixation, and after standing at room temperature for 5 min, move the cell plate to a refrigerator at 4 °C for 1 h, so that the suspended cells can be fixed at the bottom of the culture well. Pour off the fixative, wash each duplicate well 5 times with deionized water, air dry, and dry completely. Add 50 μl of 0.4% SRB staining solution prepared with 1% acetic acid to each well, and after 20-30 min at room temperature in the dark, discard the staining solution and wash 5 times with 1% acetic acid to remove unbound dye. After air drying, dissolve with 10 mmol/L (pH 10.5) 150 μl/well unbuffered Tris base. After standing at room temperature for 5 minutes, place the cell plate on a shaker for 5 minutes, which will improve the mixing of the dye. An automatic microplate reader can be used to measure the absorbance of the control group and the experimental group respectively at 565 nm and 690 nm wavelengths. Calculate the cell inhibition rate of each concentration according to the formula: cell inhibition rate=1-(OD value of experimental group/OD value of control group)×100%.

The experimental results show that the folic acid-mediated multifunctional gold nanostars of the present invention can be used in the photothermal and photokinetic combined anti-tumor drug delivery system, which can actively target tumor cells or tissues, reduce its side effects on surrounding tissues, and play a higher role. antitumor efficacy.

4. Using laser irradiation of folic acid-mediated multifunctional gold nanostars of the present invention for photothermal and photokinetic combined anti-tumor drug delivery system to measure the growth activity of tumor cells:

In vitro anti-tumor activity experiment of multifunctional gold nanostars mediated by folic acid irradiated by near-infrared laser for combined photothermal and photokinetic anti-tumor drug delivery system: MCF-7 breast cancer cells (provided by Shanghai Cell Bank) were used as Cancer cells to be investigated. Human breast cancer cells MCF-7 were cultured in RPMI1640 medium containing 10% fetal bovine serum and 1% double antibody, and incubated in a cell culture incubator at 37°C containing 5% CO ₂ , and the medium was changed every other day. Digest and passage once in 3 ^to 4 days with 0.25% trypsin (containing 0.02% EDTA), and perform relevant experiments when the cells grow to 70% ^to 80%. Cells in logarithmic growth phase were used in all experiments. The toxic effect of folic acid-mediated multifunctional gold nanostars used in photothermal and photokinetic combined anti-tumor drug delivery system on MCF-7 cells was detected by SRB method. Take the cells in the logarithmic growth phase, routinely digest them into a single-cell suspension, and count them. Adjust the density of the cells to be tested to 5×10 ³ cells/well (the edge wells are filled with sterile PBS), spread 96-well plates, and add 200 μl of culture to each well. cultured in a cell incubator at 37°C with 5% CO ₂ . After all the cells adhered to the wall for 24 hours, the original medium was discarded, and the experimental group was added with a concentration gradient of (0.5, 1, 5, 10, 50, 100 μg/mL) culture medium diluted with gold nanostars as the carrier folic acid targeting antibody For the drug delivery system of the tumor, set 3 ^to 5 replicate wells for each concentration, add blank medium to the blank cell group, set 3 ^to 5 replicate wells, as the control group, and continue to culture in the incubator. 4 hours after adding the drug in the laser group, irradiate with 808nm laser (2W/cm ² , 2min). After the light is over, place the cell culture plate in the incubator to continue culturing for 24 hours, then terminate the culture, and gently add 25 μl of pre-cooled to the surface of the culture medium. 50% TCA was fixed, and after standing at room temperature for 5 minutes, the cell plate was moved to a refrigerator at 4°C for 1 hour, so that the suspended cells could be fixed at the bottom of the culture well. Pour off the fixative, wash each duplicate well 5 times with deionized water, air dry, and dry completely. Add 50 μl of 0.4% SRB staining solution prepared with 1% acetic acid ^to each well, keep away from light for 20-30 minutes at room temperature, discard the staining solution, and wash 5 times with 1% acetic acid to remove unbound dye. After air drying, dissolve with 10mmol/L (pH 10.5) 150μl/well unbuffered Tris base. After standing at room temperature for 5 minutes, place the cell plate on a shaker for 5 minutes, which will improve the mixing of the dye. An automatic microplate reader can be used to measure the absorbance of the control group and the experimental group respectively at the wavelengths of 565nm and 690nm. Calculate the cell inhibition rate of each concentration according to the formula: cell inhibition rate=1-(OD value of experimental group/OD value of control group)×100%.

The experimental results show that the multifunctional gold nanostar mediated by folic acid of the present invention can be used in the photothermal and photokinetic combined anti-tumor drug delivery system, which can load the drug into the interior of tumor cells, and exert a good curative effect on anti-tumor drugs, and combined with 808 nm laser After that, it can inhibit the proliferation of tumor cells more obviously.

5. Determination of in vivo anti-tumor activity of folic acid-mediated multifunctional gold nanostars used in photothermal and photokinetic combined anti-tumor drug delivery systems in the present invention:

MCF-7 cells were inoculated subcutaneously under the right anterior axilla of BALB/c nude mice at 1×10 ⁷ cells/mouse. Seven days after nude mice were inoculated with tumors, 36 nude mice with tumor volume ≥ 100 mm ³ were randomly divided into 6 groups, 6 mice in each group. The specific groups are as follows: (1) control group (normal saline group); (2) normal saline laser group; (3) targeted drug group; (4) targeted drug laser group; (5) non-targeted drug group; 6) Non-targeted drug laser group. All the 6 groups were administered by tail vein, and the laser group irradiated the tumor site 4 hours after the administration, using an 808nm near-infrared light source with a power of 2W/cm ² , and the irradiation time was 1 min. Administer once every other day, inject 200 μl of physiological saline or drug solution or 100 μg/mL preparation solution each time, and administer 3 times in total. During the whole experiment, the living conditions of the nude mice were observed daily, their body weight was weighed every other day, and the long diameter (A) and short diameter (B) of the nude mouse tumors were measured with a vernier caliper, according to the formula V=1/2 (A×B ² ) to calculate the tumor volume.

Experiments have proved that under near-infrared laser irradiation, the multifunctional gold nanostars mediated by folic acid of the present invention can be used in the drug delivery system of combined photothermal and photokinetic anti-tumor drug delivery, which can significantly inhibit the increase of tumor volume in nude mice.

6. In vivo live imaging of folic acid-mediated multifunctional gold nanostars used in photothermal and photokinetic combined anti-tumor drug delivery systems:

Imaging of mice using folic acid-mediated multifunctional gold nanostars for combined photothermal and photokinetic antitumor drug delivery system (GNSTs/DOX-PEG/PEI-FA-cy5.5): Mice were treated with chloral hydrate After anesthesia, inject 5.0 μL/g body weight of cy5.5-NHS (50 μg/mL) GNSTs/DOX-PEG/PEI-FA-cy5.5 solution into the tail vein of the mouse, using a small animal in vivo imaging system Near-infrared fluorescence imaging was performed under 808 nm laser irradiation. Then cy5.5-NHS (5.0 μL/g, 50 μg/mL) and GNSTs/DOX-PEG/PEI-FA-cy5.5 (5.0 μL/g, 50 μg/mL) solutions were injected into different tail veins respectively. At different time points, the near-infrared fluorescence imaging of two groups of nude mice was observed.

According to the analysis of the results, the mouse injected with GNSTs/DOX-PEG/PEI-FA-cy5.5 solution, after 1 minute, the surface skin of the mouse emitted a stronger fluorescent signal than the control group injected with cy5.5-NHS, and the heart part An obvious fluorescent signal was emitted, indicating that the GNSTs/DOX-PEG/PEI-FA-cy5.5 solution was easy to enter blood vessels, and easily entered the mouse heart. When GNSTs/DOX-PEG/PEI-FA-cy5.5 solution was injected for 5 minutes, the fluorescence signal on the body surface weakened and the signal on the liver increased. After 4.5 hours of injection of GNSTs/DOX-PEG/PEI-FA-cy5.5 solution, the fluorescence signal in the liver area disappeared, and only part of the signal remained in the heart, and the rest of the GNSTs/DOX-PEG/PEI-FA-cy5.5 solution entered Intestine, while the fluorescent signals of mice in the cy5.5-NHS group were all transferred to the intestine. The above results show that after injection of GNSTs/DOX-PEG/PEI-FA-cy5.5 solution in mice, it is easier to enter the heart and blood vessels, and the blood vessels in the tumor site are more abundant than normal tissues, which is suitable for in vivo imaging of tumors. It has laid a good foundation and demonstrated great application value.

Compared with the prior art, the present invention has the following outstanding beneficial technical effects:

(1) In the present invention, the small molecule folic acid with good biocompatibility and tumor targeting is selected as the modified molecule, and the amino group is used as the connecting arm to modify the gold nanostar with a simple, economical and easy-to-achieve industrial production method, It will not destroy the characteristics of the gold nanostar itself, has low toxicity to organisms, and has good physical and chemical stability. It can be placed in an aqueous solution at 4°C for three months, and its particle size does not change significantly. The conditions are mild, the reaction is simple, and the yield is high, with an average of 5L synthesized at one time.

(2) The folic acid-mediated multifunctional gold nanostar provided by the present invention is used in a photothermal and photokinetic combined anti-tumor drug delivery system, as a good thermosensitivity agent for anti-tumor therapy. In the case of laser irradiation, the folic acid-mediated multifunctional gold nanostar provided by the present invention can be used in the photothermal and photodynamic combined anti-tumor drug delivery system, which can well inhibit the growth and development of tumor cells and tissues, and the inhibition rate At 70%~80% (the concentration of gold is 0.5~1mg/kg, the concentration of doxorubicin is 5~10mg/kg), and it has very little toxic and side effects on normal cells and tissues.

(3) The folic acid-mediated multifunctional gold nanostar provided by the present invention is used in a photothermal and photokinetic combined anti-tumor drug delivery system. As a good photosensitizer, it can generate active oxygen under near-infrared light irradiation, And it can show obvious cytotoxicity, combined with anti-tumor drugs can also play a more excellent anti-tumor effect of hyperthermia, phototherapy and chemotherapy.

4) The folic acid-mediated multifunctional gold nanostar provided by the present invention is used in a photothermal and photokinetic combined anti-tumor drug delivery system, which can prolong the circulation time of near-infrared fluorescent dyes in the body (up to 24-48 h) and increase the fluorescence stability. In this way, the observation window time for the detection object is extended, and the observation information is fully obtained, so that it has the potential of in vivo imaging. It is an innovation in anti-tumor drugs and has huge economic and social benefits.

Claims (9)

1. A preparation method of a photothermal and photodynamic combined anti-tumor drug delivery system using folic acid-mediated gold nanostars as a carrier, characterized in that the surface of the gold nanostars is linked to a reactive amino group by a covalent bond and the carboxyl group, and then connect the targeting molecule through an amide reaction to obtain an active targeting gold nanostar solution; then chemically react the amino group at the end of the targeting compound with a near-infrared fluorescent probe to obtain a folic acid-targeting and near-infrared fluorescent probe. Imaged gold nanostar solution; then the anti-tumor drug doxorubicin is directly loaded on the surface of gold nanostars through electrostatic adsorption, that is, folic acid-mediated multifunctional gold nanostars are used for combined photothermal and photokinetic anti-tumor drug delivery system, the mass ratio of doxorubicin to gold nanostars is 1.5-3:1, and the specific preparation method is as follows: (1) Preparation of aminated gold nanostars: Add 10-20 mL of a gold nanostar solution with a concentration of 50 μg/mL at 18-25°C and a magnetic stirring condition of 300-720 r/min, and then add a concentration of 0.1- 1-5 mL of 10.0 mg/mL amino compound was stirred in the dark for 6-12 h, then centrifuged at 10,000 rpm for 30 min at high speed, and then reconstituted with ultrapure water to obtain an aminated gold nanostar solution; The amino compound is one or a mixture of NH ₂ -PEG2000-SH and PEI-SH; (2) Preparation of targeted gold nanostars: Weigh 15-20 mg of folic acid, 10-15 mg of N, N'-dicyclohexylcarbimide, and 7.5-10.5 mg of N-hydroxysuccinimide, and then Add 2.5-5.0 mL of the reaction solution in turn, mix and dissolve, and then activate the carboxyl group, that is, at 40-50 ° C, 300-720 r/min, avoid light and magnetically stir the reaction for 8-24 hours, and then centrifuge the obtained solution at 10,000 rpm for 30 minutes The precipitate was obtained, and the precipitate was redissolved with ultrapure water, added to the aminated gold nanostar solution prepared in step (1) for acylation reaction, stirred and reacted at room temperature in the dark for 12-24 hours, and then centrifuged at 10000 rpm for 30 minutes at high speed to remove free small Molecules, to obtain folic acid-targeted gold nanostar solution containing amino groups at the end; Described reaction solution is a kind of in dimethyl sulfoxide, phosphate buffer saline and acetone; (3) Preparation of gold nanostar solution with near-infrared fluorescence imaging and targeting: Dissolve 0.01-2 mg fluorescent probe in 0.01-1 mL of reaction solvent, add folic acid-targeted gold nanostars prepared in step (2) Carry out amide reaction in the star solution, stir and react at room temperature in the dark for 12-24 hours, then ultracentrifuge at 10,000rpm for 30 minutes to obtain a precipitate, and redissolve the precipitate in 0.01-1mL of ultrapure water to obtain gold nanostars with targeting properties and near-infrared fluorescence. Imaging anti-tumor drug delivery systems; The fluorescent probe is one of indole-5-cyanine, indole-5.5-cyanine and indole-5.5-cyanine-N-hydroxysuccinimide; Described reaction solvent is a kind of in dimethyl sulfoxide, phosphate buffer saline and acetone; (4) Preparation of doxorubicin-loaded gold nanostar solution: the targeted gold nanostar solution with near-infrared fluorescence imaging prepared in step (3) was dried to obtain a powder, and the powder and doxorubicin were mixed at a mass ratio of 1:1.5 Add ～3.0 to 10～40 mL of reaction solvent in turn, under the protection of nitrogen, ultrasonically dissolve, and stir at 18～25°C at 300～720r/min in the dark for 24～48h to obtain a mixed solution, and then mix the obtained The solution was ultracentrifuged at 10,000rpm for 30 minutes to obtain a precipitate, which was redissolved in ultrapure water and dried to obtain a drug delivery system using folic acid-mediated gold nanostars as a carrier for combined photothermal and photokinetic anti-tumor effects; The drying is one of freeze drying, vacuum drying and drying. 2. The preparation method of the photothermal and photodynamic combined anti-tumor drug delivery system with folic acid-mediated gold nanostars as the carrier according to claim 1, characterized in that, it is realized by the following steps: (1) Preparation of aminated gold nanostar solution: Add 20 mL of gold nanostar solution with a concentration of 50 μg/mL, and then add NH ₂ -PEG2000 with a concentration of 1 mg/mL under the condition of 25°C and 540 r/min magnetic stirring -SH solution 1mL, stirred at room temperature and protected from light for 12h, then centrifuged at 10000rpm for 30min at high speed to obtain a precipitate, which was redissolved in 21mL of ultrapure water to obtain an aminated gold nanostar solution; (2) Preparation of targeted gold nanostars: Weigh 19.91 mg of folic acid, 13.94 mg of N,N'-dicyclohexylcarboimide, and 10.36 mg of N-hydroxysuccinimide, and then add 4 mL of di In methyl sulfoxide, mix and dissolve, and then activate the carboxyl group, that is, at 50°C and 480r/min to avoid light and magnetically stir for 12h, then centrifuge the resulting solution at 10,000rpm for 30min at high speed to obtain a precipitate, and redissolve the precipitate with ultrapure water. Add the aminated gold nanostar solution prepared in step (1) for acylation reaction, stir at room temperature in the dark for 24 hours, then centrifuge at 10,000 rpm for 30 minutes to remove free small molecules, and obtain folic acid-targeted gold nanostars containing amino groups at the end solution; (3) Preparation of gold nanostar solution with near-infrared fluorescence imaging and targeting: Dissolve 1 mg of fluorescent probe indole-5.5-cyanine-N-hydroxysuccinimide in 1 mL of dimethyl sulfoxide, add The folic acid prepared in step (2) was targeted to the gold nanostar solution for amide reaction, stirred at room temperature for 24 hours in the dark, and then ultracentrifuged at 10,000 rpm for 30 minutes to obtain a precipitate, which was redissolved with 0.5 mL of ultrapure water, and the gold nanostar was used as a carrier with Near-infrared fluorescence imaging and targeting of gold nanostar solutions; (4) Preparation of gold nanostar solution loaded with doxorubicin: the gold nanostar solution with near-infrared fluorescence imaging and targeting prepared in step (3) was freeze-dried to obtain a powder, and the powder and doxorubicin were mixed in a mass ratio Add 1:2 to 24mL dimethyl sulfoxide sequentially, under the protection of nitrogen, ultrasonically dissolve, and stir at 25°C at 480r/min in the dark for 24h to obtain a mixed solution, and then ultracentrifuge the obtained mixed solution at 10000rpm After 30 minutes, the precipitate was obtained, and the precipitate was redissolved with ultrapure water, and freeze-dried to obtain a drug delivery system powder with folic acid-mediated gold nanostar as a carrier for combined photothermal and photokinetic anti-tumor. 3. The preparation method of the drug delivery system based on folic acid-mediated gold nanostars as a carrier of photothermal and photodynamic combined anti-tumor according to claim 1, characterized in that, it is realized by the following steps: (1) Preparation of aminated gold nanostars: Add 15 mL of gold nanostar solution with a concentration of 50 μg/mL, and then add 3 mL of PEI-SH with a concentration of 5 mg/mL at 20 °C and 600 r/min magnetic stirring conditions, Stir the reaction in the dark for 8 hours, then centrifuge at a high speed of 10000 rpm for 30 minutes, and then redissolve with ultrapure water to obtain an aminated gold nanostar solution; (2) Preparation of targeted gold nanostars: Weigh 18 mg of folic acid, 12 mg of N, N'-dicyclohexylcarbimide, and 9 mg of N-hydroxysuccinimide, and then add 4 mL of phosphate buffer in sequence In, mix and dissolve, and then activate the carboxyl group, that is, at 45°C, 500r/min, shielded from light and magnetically stirred for 16h, then centrifuge the resulting solution at 10,000 rpm for 30min to obtain a precipitate, redissolve the precipitate with ultrapure water, and add the step ( 1) Acylation reaction was carried out in the prepared aminated gold nanostar solution, stirred at room temperature in the dark for 16 hours, then centrifuged at 10,000 rpm for 30 minutes to remove free small molecules, and a folic acid-targeted gold nanostar solution containing amino groups at the end was obtained; (3) Preparation of gold nanostar solution with near-infrared fluorescence imaging and targeting: Dissolve 1.5 mg of fluorescent probe indole-5-cyanine in 0.8 mL of phosphate buffer, add folic acid prepared in step (2) Carry out amide reaction in the targeting gold nanostar solution, stir and react at room temperature in the dark for 16 hours, then ultracentrifuge at 10,000rpm for 30min to obtain a precipitate, and redissolve the precipitate in 0.8mL of ultrapure water to obtain a gold nanostar with targeting and near-infrared properties. Fluorescence imaging anti-tumor drug delivery system; (4) Preparation of doxorubicin-loaded gold nanostar solution: The targeted gold nanostar solution with near-infrared fluorescence imaging prepared in step (3) was vacuum-dried to obtain a powder, and the powder and doxorubicin were mixed in a mass ratio of 1: 2.5 Add it to 35mL of phosphate buffer in turn, dissolve it under nitrogen protection, ultrasonically dissolve, stir and react at 600r/min in the dark for 30h at 20°C to obtain a mixed solution, and then ultracentrifuge the obtained mixed solution at 10000rpm for 30min to obtain Precipitate, redissolve the precipitate with ultra-pure water, and vacuum-dry to obtain a drug delivery system powder with folic acid-mediated gold nanostars as the carrier for combined photothermal and photokinetic anti-tumor. 4. The preparation method of a drug delivery system based on folic acid-mediated gold nanostars as a carrier of photothermal and photodynamic combined anti-tumor according to claim 1, characterized in that, it is realized by the following steps: (1) Preparation of aminated gold nanostars: Add 15 mL of gold nanostar solution with a concentration of 50 μg/mL, and then add 3 mL of amino compounds with a concentration of 5.0 mg/mL under the condition of 21 ° C and 510 r/min magnetic stirring, Stir the reaction in the dark for 9 hours, then centrifuge at a high speed of 10000 rpm for 30 minutes, and then redissolve with ultrapure water to obtain an aminated gold nanostar solution; (2) Preparation of targeted gold nanostars: Weigh 17.5 mg of folic acid, 12.5 mg of N, N'-dicyclohexyl carboximide, and 9 mg of N-hydroxysuccinimide, and then add 3.5 mL of them in turn to react In the solution, mix and dissolve, and then activate the carboxyl group, that is, at 45°C and 510r/min to avoid light and magnetically stir for 16h, then centrifuge the resulting solution at 10,000 rpm for 30min to obtain a precipitate, which is redissolved with ultrapure water, and added to the step (1) Acylation reaction was carried out in the prepared aminated gold nanostar solution, stirred at room temperature in the dark for 18 hours, then centrifuged at 10,000 rpm for 30 minutes to remove free small molecules, and a folic acid-targeted gold nanostar solution containing amino groups at the end was obtained; (3) Preparation of gold nanostar solution with near-infrared fluorescence imaging and targeting: Dissolve 1 mg of fluorescent probe in 0.5 mL of reaction solvent, add folic acid targeting gold nanostar solution prepared in step (2) for amide Reaction, stirring at room temperature and avoiding light for 18 hours, and then ultracentrifuging at 10,000 rpm for 30 minutes to obtain a precipitate, which was redissolved in 0.5 mL of ultrapure water to obtain an anti-tumor drug delivery system using gold nanostars as a carrier with targeting and near-infrared fluorescence imaging; (4) Preparation of doxorubicin-loaded gold nanostar solution: the targeted gold nanostar solution with near-infrared fluorescence imaging prepared in step (3) was dried to obtain a powder, and the powder and doxorubicin were mixed at a mass ratio of 1:1.7 Sequentially add to 25mL reaction solvent, under the protection of nitrogen, ultrasonically dissolve, stir and react at 510r/min in the dark for 36h at 21°C to obtain a mixed solution, and then ultracentrifuge the obtained mixed solution at 10000rpm for 30min to obtain a precipitate. Reconstituted with ultra-pure water and dried to obtain a drug delivery system powder with folic acid-mediated gold nanostars as a carrier for combined photothermal and photokinetic antitumor effects. 5. The preparation method of a drug delivery system based on folic acid-mediated gold nanostars as a carrier of photothermal and photodynamic combined anti-tumor according to claim 1, characterized in that, it is realized by the following steps: (1) Preparation of aminated gold nanostars: Add 13-17mL of gold nanostar solution with a concentration of 50μg/mL at 20-22°C and magnetic stirring at 500-600r/min, and then add 6-8mg /mL amino compound 2-3mL, stirred and reacted in the dark for 8-10 h, then centrifuged at 10,000 rpm for 30 min, and then re-dissolved with ultrapure water to obtain an aminated gold nanostar solution; (2) Preparation of targeted gold nanostars: Weigh 17-18 mg of folic acid, 12-13 mg of N, N'-dicyclohexylcarbimide, and 8-9.5 mg of N-hydroxysuccinimide, and then Add 3-4mL reaction solution in turn, mix and dissolve, and then activate the carboxyl group, that is, at 43-47°C, 500-600r/min, avoid light and magnetically stir for 12-20h, and then centrifuge the resulting solution at 10,000 rpm for 30min to obtain Precipitate, redissolve the precipitate with ultrapure water, add the aminated gold nanostar solution prepared in step (1) for acylation reaction, stir at room temperature in the dark for 16-20h, then centrifuge at 10,000 rpm for 30min at high speed to remove free small molecules , to obtain a folic acid-targeted gold nanostar solution containing an amino group at the end; (3) Preparation of gold nanostar solution with near-infrared fluorescence imaging and targeting: Dissolve 0.8-1.2 mg of fluorescent probe in 0.4-0.6 mL of reaction solvent, add folic acid-targeted gold nanoparticles prepared in step (2) Carry out amide reaction in the star solution, stir the reaction for 16-20 hours at room temperature in the dark, and then ultracentrifuge at 10,000 rpm for 30 minutes to obtain a precipitate. Fluorescence imaging anti-tumor drug delivery system; (4) Preparation of doxorubicin-loaded gold nanostar solution: the targeted gold nanostar solution with near-infrared fluorescence imaging prepared in step (3) was dried to obtain a powder, and the powder and doxorubicin were mixed at a mass ratio of 1:1.8 -2.2 Add to 20-30mL reaction solvent in turn, under the protection of nitrogen, ultrasonically dissolve, and stir at 500-600r/min in the dark for 30-40h at 20-22°C to obtain a mixed solution, and then mix the obtained mixed solution Ultracentrifuge at 10,000rpm for 30min to obtain a precipitate, redissolve the precipitate in ultrapure water, and dry to obtain a drug delivery system powder with folic acid-mediated gold nanostars as a carrier for combined photothermal and photodynamic antitumor effects. 6. The photothermal and photokinetic combined anti-tumor drug delivery system mediated by folic acid-mediated gold nanostars prepared by the method of any one of claims 1 or 2-5 is used in the preparation of active targeting anti-tumor drug delivery carriers Applications. 7. The photothermal and photodynamic combined anti-tumor drug delivery system with folic acid-mediated gold nanostars as the carrier prepared by the method described in any one of claims 1 or 2-5 is used in the preparation of anti-tumor photodynamic or photothermal therapy Applications in thermosensitizers. 8. The photothermal and photodynamic combined anti-tumor drug delivery system mediated by folic acid-mediated gold nanostars prepared by the method of any one of claims 1 or 2-5 triggers chemical drugs under the preparation of near-infrared laser irradiation Application of targeted rapid release drug in tumor tissue. 9. The photothermal and photodynamic combined anti-tumor drug delivery system with folic acid-mediated gold nanostars as the carrier prepared by the method of any one of claims 1 or 2-5 is used in the preparation of photothermal and photodynamic combined anti-tumor The application of the drug delivery system in the preparation of in vivo fluorescence imaging drugs.