The application discloses an anti-metastasis nanodrug as well as a preparation method and application thereof and a chemotherapy-photodynamic combined anti-metastasis diagnosis and treatment reagent.

Abstract

The application belongs to the technical field of anticancer drugs, and particularly relates to an anti-metastasis nano drug and a preparation method and application thereof, and a chemotherapy-light dynamic combined anti-metastasis diagnosis and treatment reagent. The anti-metastasis nano drug provided by the application comprises vitamin E polyethylene glycol succinate nanomicelles, and lauric acid modified tetravalent platinum prodrugs, all-trans retinoic acid and hydrophobic photosensitizers coated in the vitamin E polyethylene glycol succinate nanomicelles. After the anti-metastasis nano drug provided by the application is passively targeted to tumor cells through the EPR effect, the hydrophobic photosensitizer can increase the generation of singlet oxygen under the irradiation of red light, and cooperates with the chemotherapy drug (tetravalent platinum) to achieve the combination of chemotherapy and photodynamic therapy, so that the drug resistance can be overcome and good treatment effect can be achieved. Meanwhile, the anti-metastasis nano drug provided by the application has strong cytotoxicity, exhibits excellent anti-metastasis effect, and can be used for anti-metastasis tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of anticancer drugs, and specifically relates to an anti-metastasis nano drug, a preparation method and application thereof, and a chemotherapy-photodynamic combined anti-metastasis diagnostic and therapeutic reagent. Background Art

[0002] Tumor metastasis, the gradual development of secondary tumors at sites far from the primary tumor, is one of the most serious challenges in cancer treatment. Currently, more than 500 related anti-metastatic agents have been studied and reported. The most studied anti-metastatic drugs mainly include angiogenesis inhibitors and matrix metalloproteinase inhibitors. Anti-metastatic drugs approved for clinical use by the U.S. Food and Drug Administration (FDA) also belong to these two categories. However, these anti-metastatic drugs have almost no effect on patients with advanced disease, especially in advanced stage patients, due to their non-selective molecular inhibition and lack of significant improvement in patient survival. As a result, the anti-metastatic effects of currently used anti-metastatic drugs are relatively weak.

[0003] All-trans retinoic acid (ATRA), a metabolite of vitamin A, is a first-line treatment for leukemia and has been extensively studied due to its efficacy in treating other solid cancers. It promotes cell differentiation, exhibits low toxicity, regulates cancer cell plasticity and motility, and inhibits tumor metastasis. It is widely used in the treatment of cancers such as squamous cell carcinoma and melanoma. However, due to various drawbacks, such as its short half-life, poor water solubility, and susceptibility to inactivation by light, oxygen, and heat, its efficacy in treating solid tumors is relatively poor.

[0004] Currently, nanocarriers are often used to increase their water solubility and stability, significantly extending their in vivo circulation time. Furthermore, due to the high permeability and retention effect (EPR effect) of solid tumors, anti-metastatic nanoparticles can be passively targeted and accumulated in tumor tissues. To date, there have been reports of using liposomes, polymers, and other materials loaded with ATRA for cancer treatment. However, the therapeutic effect of anti-tumor metastasis remains poor. Summary of the Invention

[0005] The purpose of the present invention is to provide an anti-metastatic nanomedicine, its preparation method and application, and a chemotherapy-photodynamic combined anti-metastatic diagnostic and therapeutic reagent. The anti-metastatic nanomedicine provided by the present invention is relatively uniformly dispersed in water and has good stability. It not only has good chemotherapy and photodynamic therapy effects, but also has strong cytotoxicity, showing excellent anti-metastatic effects.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides an anti-metastasis nanomedicine, comprising vitamin E polyethylene glycol succinate nanomicelles, and a lauric acid-modified tetravalent platinum prodrug, all-trans retinoic acid and a hydrophobic photosensitizer encapsulated in the vitamin E polyethylene glycol succinate nanomicelles; the lauric acid-modified tetravalent platinum prodrug is obtained by reacting Pt(IV)(OH)2 and lauric anhydride.

[0008] Preferably, the hydrophobic photosensitizer includes m-tetraphenylporphine and / or hematophorin methyl ether.

[0009] Preferably, the particle size of the anti-metastasis nanomedicine is 60 nm.

[0010] Preferably, the preparation method of the lauric acid-modified tetravalent platinum prodrug comprises the following steps:

[0011] Mixing lauric anhydride, a complex [Pt(NH2)2(OH)2Cl2], a first organic solvent, and a second organic solvent for a grafting reaction to obtain a grafting reaction liquid; the first organic solvent is an organic solvent miscible with water, and the second organic solvent is an organic solvent immiscible with water;

[0012] The grafting reaction solution is removed from the second organic solvent and then mixed with water, and the lauric acid-modified tetravalent platinum prodrug is obtained after solid-liquid separation.

[0013] The present invention provides a method for preparing the anti-metastasis nanomedicine described in the above technical solution, comprising the following steps:

[0014] mixing a lauric acid-modified tetravalent platinum prodrug, all-trans retinoic acid, a hydrophobic photosensitizer, and a third organic solvent to obtain a drug solution;

[0015] dissolving vitamin E polyethylene glycol succinate in water to obtain a vitamin E polyethylene glycol succinate solution;

[0016] mixing the drug solution and the vitamin E polyethylene glycol succinate solution to obtain a mixed liquid;

[0017] The mixed material liquid is added dropwise to an inorganic strong alkali aqueous solution and mixed, and then the third organic solvent is removed to obtain the anti-metastasis nano drug dispersion.

[0018] Preferably, the molar ratio of the lauric acid-modified tetravalent platinum prodrug to the hydrophobic photosensitizer is 0.0025:0.01.

[0019] Preferably, the molar ratio of the lauric acid-modified tetravalent platinum prodrug to all-trans retinoic acid is 0.005:0.5.

[0020] Preferably, the mass ratio of the lauric acid-modified tetravalent platinum prodrug to vitamin E polyethylene glycol succinate is 3.5:7.5.

[0021] The present invention provides the use of the anti-metastasis nanomedicine described in the above technical solution or the anti-metastasis nanomedicine prepared by the preparation method described in the above technical solution in the preparation of anticancer drugs.

[0022] The present invention provides a chemotherapy-photodynamic combined anti-metastasis diagnostic and therapeutic reagent, which includes the anti-metastasis nanomedicine described in the above technical solution or the anti-metastasis nanomedicine prepared by the preparation method described in the above technical solution.

[0023] The present invention provides an anti-metastasis nanomedicine, comprising vitamin E polyethylene glycol succinate nanomicelles, and a lauric acid-modified tetravalent platinum prodrug, all-trans retinoic acid (ATRA), and a hydrophobic photosensitizer encapsulated in the vitamin E polyethylene glycol succinate nanomicelles; the lauric acid-modified tetravalent platinum prodrug is obtained by reacting Pt(IV)(OH)2 and lauric anhydride. The present invention uses kinetically stable core-shell structured nanomicelles formed by vitamin E polyethylene glycol succinate (vitamin E-TPGS or TPGS) as a drug carrier, and has a good encapsulation effect on a lauric acid-modified tetravalent platinum prodrug, ATRA, and a hydrophobic photosensitizer. The encapsulation is good and the drug loading capacity is high, thereby achieving the co-transport of cisplatin prodrug, ATRA, and photosensitizer. The anti-metastatic nanomedicine provided by the present invention is passively and targetedly transported into tumor cells through the EPR effect. The hydrophobic photosensitizer can increase the production of singlet oxygen under the irradiation of red light, and synergistically acts with the chemotherapy drug (tetravalent platinum). Chemotherapy and photodynamic therapy are combined, which can overcome drug resistance and achieve good therapeutic effects. At the same time, the anti-metastatic nanomedicine provided by the present invention has strong cytotoxicity, exhibits excellent anti-metastatic effects, and can be used for anti-metastatic tumor treatment.

[0024] Furthermore, in the present invention, the preparation method of the lauric acid-modified tetravalent platinum prodrug includes the following steps: mixing lauric anhydride, a complex [Pt(NH2)2(OH)2Cl2], a first organic solvent, and a second organic solvent to perform a grafting reaction to obtain a grafting reaction liquid; the first organic solvent is an organic solvent miscible with water, and the second organic solvent is an organic solvent immiscible with water; removing the second organic solvent from the grafting reaction liquid, mixing it with water, and performing solid-liquid separation to obtain the lauric acid-modified tetravalent platinum prodrug. In the present invention, the cisplatin prodrug is readily soluble in water and is therefore not easily supported by the vitamin E polyethylene glycol succinate nanomicelles. The present invention modifies the complex [Pt(NH2)2(OH)2Cl2] by using lauric anhydride, chemically bonds lauric anhydride to the complex [Pt(NH2)2(OH)2Cl2] molecule, and enhances the hydrophobicity of the cisplatin prodrug. The obtained lauric acid-modified tetravalent platinum prodrug can be effectively coated by nanomicelles and has good encapsulation and high drug loading capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of Pt-C12 prepared in Example 1;

[0026] Figure 2 The SEM and TEM images of Pt-ATRA-TPP@TPGS prepared in Example 1;

[0027] Figure 3 The UV absorption spectra, drug release curves, particle size distributions of TPP, ATRA, and Pt-ATRA-TPP@TPGS in Example 1, as well as the zeta potential of Pt-ATRA-TPP@TPGS at different pH values ​​are shown;

[0028] Figure 4 For RNO / His reagent detection and different drugs in Example 1 633nm (30mW / cm 3 ) produced under light 1 O2, Figure 4 The A in it is TPP dark, Figure 4 B in the figure is TPP illumination. Figure 4 C in the figure is Pt-ATRA-TPP@TPGS dark, Figure 4 D in the figure is the illumination of Pt-ATRA-TPP@TPGS;

[0029] Figure 5 Methylene blue bleaching method for detecting hydroxyl radicals, Figure 5 The A in it is TPP dark, Figure 5 B in the figure is TPP illumination. Figure 5 C in the figure is Pt-ATRA-TPP@TPGS dark, Figure 5 D in the figure is the illumination of Pt-ATRA-TPP@TPGS;

[0030] Figure 6 This is the result of intracellular singlet oxygen detection;

[0031] Figure 7 The results of cytotoxicity assay were obtained by MTT method. Figure 7 A in the figure represents A549 cells. Figure 7 B in the figure is A549R cells;

[0032] Figure 8 The live and dead cell staining results under the treatment of each drug under dark conditions;

[0033] Figure 9 Live and dead cell staining results of TPP and Pt-ATRA-TPP@TPGS under light and dark conditions;

[0034] Figure 10 is the scratch test result;

[0035] Figure 11 This is the result of Transwell anti-metastasis experiment;

[0036] Figure 12 This is a mouse melanoma lung metastasis model;

[0037] Figure 13 H&E staining of the lungs in a mouse melanoma lung metastasis model;

[0038] Figure 14 The tumor inhibition effect of Pt-ATRA-TPP@TPGS;

[0039] Figure 15 H&E staining results of heart, liver, spleen, lung, kidney, and tumor;

[0040] Figure 16 TUNEL staining results of tumors;

[0041] Figure 17 Synthesis route of lauric acid-modified tetravalent platinum prodrug Pt-C12. DETAILED DESCRIPTION

[0042] The present invention provides an anti-metastasis nanomedicine, comprising vitamin E polyethylene glycol succinate nanomicelles, and a lauric acid-modified tetravalent platinum prodrug, all-trans retinoic acid and a hydrophobic photosensitizer encapsulated in the vitamin E polyethylene glycol succinate nanomicelles; the lauric acid-modified tetravalent platinum prodrug is obtained by reacting Pt(IV)(OH)2 and lauric anhydride.

[0043] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0044] The anti-metastasis nano drug provided by the invention comprises vitamin E polyethylene glycol succinate nano micelles.

[0045] The chemical structure of vitamin E polyethylene glycol succinate provided by the present invention is shown in Formula 1:

[0046]

[0047] Vitamin E polyethylene glycol succinate (Vitamin E-TPGS or TPGS) provided by the present invention is a nonionic amphiphile synthesized by esterifying vitamin E and succinate. TPGS can spontaneously form kinetically stable core-shell micelles (12-15 nm in diameter) and exhibits properties as a solubilizer, emulsifier, dispersant, and gelling agent. Its aggregation behavior can be modulated in combination with other amphiphiles and organic additives.

[0048] In aqueous solution, when TPGS reaches a certain concentration, the molecules self-assemble to form orderly arranged micelles. The mechanism of micelle formation is that after the adsorption capacity of TPGS reaches saturation, the excess TPGS is dispersed in the aqueous solution. Due to the presence of hydrophobic groups, the repulsive force between water molecules and TPGS is stronger than the attractive force. The hydrophobic groups associate under the action of van der Waals forces to form the micelle core, while the hydrophilic groups face outward to form the micelle outer layer, which is stably dispersed in the aqueous solution.

[0049] The anti-metastasis nanomedicine provided by the present invention comprises a lauric acid-modified tetravalent platinum prodrug encapsulated in the vitamin E polyethylene glycol succinate nanomicelles. In the present invention, the lauric acid-modified tetravalent platinum prodrug is obtained by reacting Pt(IV)(OH)2 and lauric anhydride.

[0050] In the present invention, the method for preparing the lauric acid-modified tetravalent platinum prodrug preferably comprises the following steps:

[0051] Mixing lauric anhydride, a complex [Pt(NH2)2(OH)2Cl2], a first organic solvent, and a second organic solvent for a grafting reaction to obtain a grafting reaction liquid; the first organic solvent is an organic solvent miscible with water, and the second organic solvent is an organic solvent immiscible with water;

[0052] The grafting reaction solution is removed from the second organic solvent and then mixed with water, and the lauric acid-modified tetravalent platinum prodrug is obtained after solid-liquid separation.

[0053] The present invention mixes lauric anhydride, a complex [Pt(NH2)2(OH)2Cl2], a first organic solvent and a second organic solvent for grafting reaction to obtain a grafting reaction liquid; the first organic solvent is an organic solvent miscible with water, and the second organic solvent is an organic solvent immiscible with water.

[0054] In the present invention, the method for preparing lauric anhydride preferably comprises the following steps: mixing lauric acid, a dehydrating agent, and an organic solvent in a protective gas atmosphere to conduct a dehydration reaction to produce lauric anhydride. In the present invention, the dehydrating agent is preferably dicyclohexylcarbodiimide (DCC). The organic solvent is preferably dichloromethane. The molar ratio of lauric acid to the dehydrating agent is preferably 10:5.53. The present invention does not require a specific amount of the organic solvent, as long as the dehydration reaction proceeds smoothly. In the present invention, the protective gas is preferably nitrogen. The dehydration reaction temperature is preferably 25°C, and the duration is preferably 24 hours; the dehydration reaction is carried out under stirring. After the dehydration reaction, a dehydration reaction liquid is obtained. In the present invention, the dehydration reaction liquid is preferably cooled to room temperature and subjected to solid-liquid separation. The resulting solid product is washed to produce a wash supernatant; the wash supernatant is dried to produce lauric anhydride. In the present invention, the solid-liquid separation is preferably performed by suction filtration, the reagent used for washing is preferably dichloromethane; and the drying is preferably performed by rotary evaporation drying.

[0055] In the present invention, the molar ratio of the lauric anhydride to the complex [Pt(NH2)2(OH)2Cl2] is preferably 2.4:0.6.

[0056] In the present invention, the first organic solvent is preferably anhydrous dimethyl sulfoxide (DMSO). The second organic solvent is preferably chloroform. The volume ratio of the first organic solvent to the second organic solvent is preferably 10:3. The present invention has no special requirements for the total amount of the first organic solvent and the second organic solvent, as long as the grafting reaction proceeds smoothly.

[0057] In the present invention, the grafting reaction is preferably carried out at room temperature. After the grafting reaction is completed, the grafting reaction solution is preferably mixed with water and then cooled at 0-4°C. The resulting cooled solution is subjected to solid-liquid separation, and the resulting solid product is washed to obtain a lauric acid-modified tetravalent platinum prodrug. In the present invention, the volume ratio of the grafting reaction solution to water is preferably 1:10. The cooling time is preferably 0.5h. The solid-liquid separation is preferably performed by centrifugation, the centrifugation speed is preferably 5000rpm, and the time is preferably 5min. The washing is preferably performed by washing with water, ether, and acetone in sequence, with each solvent washing 2-3 times.

[0058] In the present invention, the chemical structure of the lauric acid-modified tetravalent platinum prodrug is shown in Formula 2:

[0059]

[0060] The anti-metastasis nano drug provided by the present invention comprises a hydrophobic photosensitizer coated in the vitamin E polyethylene glycol succinate nano micelle.

[0061] In the present invention, the hydrophobic photosensitizer preferably includes m-tetraphenylporphine (TPP) and / or hematophore methyl ether, more preferably m-tetraphenylporphine (TPP).

[0062] In the present invention, the chemical structure of the TPP is shown in Formula 3:

[0063]

[0064] The anti-metastasis nano drug provided by the invention comprises all-trans retinoic acid encapsulated in the vitamin E polyethylene glycol succinate nano micelles.

[0065] In the present invention, the all-trans retinoic acid (ATRA) can promote cell differentiation, has low toxicity, and can regulate the plasticity and motility of cancer cells, and has the effect of inhibiting tumor metastasis.

[0066] In the present invention, the particle size of the anti-metastasis nanomedicine is preferably 60 nm.

[0067] In the anti-metastasis nanomedicine provided by the present invention, the mass ratio of the lauric acid-modified tetravalent platinum prodrug, the hydrophobic photosensitizer and ATRA is preferably 7:18:30.

[0068] The present invention provides a method for preparing the anti-metastasis nanomedicine described in the above technical solution, comprising the following steps:

[0069] mixing a lauric acid-modified tetravalent platinum prodrug, all-trans retinoic acid, a hydrophobic photosensitizer, and a third organic solvent to obtain a drug solution;

[0070] dissolving vitamin E polyethylene glycol succinate in water to obtain a vitamin E polyethylene glycol succinate solution;

[0071] mixing the drug solution and the vitamin E polyethylene glycol succinate solution to obtain a mixed liquid;

[0072] The mixed material liquid is added dropwise to an inorganic strong alkali aqueous solution and mixed, and then the third organic solvent is removed to obtain the anti-metastasis nano drug dispersion.

[0073] The present invention combines a lauric acid-modified tetravalent platinum prodrug, all-trans retinoic acid, a hydrophobic photosensitizer, and a third organic solvent to produce a drug solution. In the present invention, the third organic solvent is preferably chloroform. In the present invention, the molar ratio of the lauric acid-modified tetravalent platinum prodrug to the hydrophobic photosensitizer is 0.0025:0.01. The molar ratio of the lauric acid-modified tetravalent platinum prodrug to all-trans retinoic acid is 0.005:0.5.

[0074] In the present invention, the mixing preferably includes the following steps: dissolving the lauric acid-modified tetravalent platinum prodrug in a portion of the third organic solvent to obtain a lauric acid-modified tetravalent platinum prodrug solution; dissolving the hydrophobic photosensitizer in another portion of the third organic solvent to obtain a photosensitizer solution; dissolving the all-trans retinoic acid in the remaining third organic solvent to obtain an all-trans retinoic acid solution; and mixing the lauric acid-modified tetravalent platinum prodrug solution, the photosensitizer solution, and the all-trans retinoic acid solution. In the present invention, the molar concentration of the lauric acid-modified tetravalent platinum prodrug solution is preferably 2.5 mmol / L; the molar concentration of the photosensitizer solution is preferably 10 mmol / L; the molar concentration of the all-trans retinoic acid solution is preferably 500 mmol / L; when the lauric acid-modified tetravalent platinum prodrug solution, the photosensitizer solution, and the all-trans retinoic acid solution are mixed, the volume ratio of the lauric acid-modified tetravalent platinum prodrug solution, the photosensitizer solution, and the all-trans retinoic acid solution is preferably 2:2:1.

[0075] The present invention dissolves vitamin E polyethylene glycol succinate in water to obtain a vitamin E polyethylene glycol succinate solution. In the present invention, the mass ratio of the lauric acid-modified tetravalent platinum prodrug to vitamin E polyethylene glycol succinate is preferably 3.5:7.5. The mass ratio of the vitamin E polyethylene glycol succinate to the volume of water is preferably 7.5 mg:5 mL.

[0076] After obtaining the drug solution and the vitamin E polyethylene glycol succinate solution, the present invention mixes the drug solution and the vitamin E polyethylene glycol succinate solution to obtain a mixed liquid.

[0077] After obtaining the mixed liquid, the present invention adds the mixed liquid dropwise to an inorganic strong alkali aqueous solution for mixing, and then removes the third organic solvent to obtain a dispersion of the anti-metastasis nanodrug. In the present invention, the inorganic strong alkali aqueous solution is preferably a sodium hydroxide aqueous solution, and the molar concentration of the inorganic strong alkali aqueous solution is preferably 1 mol / L. The molar ratio of the lauric acid-modified tetravalent platinum prodrug and the inorganic strong base is 0.5:0.55. In the present invention, the specific implementation method of removing the third organic solvent is preferably evaporation, and the evaporation is carried out under ultrasonic oscillation conditions, the evaporation temperature is preferably 40°C, the evaporation is carried out under water bath conditions, and the ultrasonic oscillation time is preferably 1 hour, until the third organic solvent is volatilized.

[0078] The dispersion liquid of the anti-metastatic nanodrug provided by the present application is preferably a water dispersion liquid of the anti-metastatic nanodrug. In the present application, the water dispersion liquid of the anti-metastatic nanodrug has a Pt concentration of 99.2 μM, a loading rate as high as 99.2%, a TPP content of 296 μM, and a loading rate of 73.88% as determined by ultraviolet-visible spectrophotometry. The ATRA content is about 1 mM, and the loading rate is 10% as determined by ultraviolet-visible spectrophotometry.

[0079] The present application provides the use of the anti-metastatic nanodrug prepared by the preparation method in the preparation of an anticancer drug.

[0080] The present application provides a chemotherapy-photodynamic combined anti-metastatic diagnosis and treatment reagent, which comprises the anti-metastatic nanodrug prepared by the preparation method.

[0081] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0082] The experimental reagents and instruments used in the following examples are shown in Tables 1 and 2.

[0083] Table 1 Experimental reagents and manufacturers

[0084]

[0085]

[0086] Table 2 Main experimental instruments

[0087] Instrument name Model Manufacturer Electronic balance BSA224S-CW Sartorius, Germany High-speed refrigerated centrifuge 5418R Eppendorf, Germany Super-clean sterile workbench SW-CJ-1FD Suzheng Antai Carbon dioxide incubator HF90 Liconbio Ultraviolet spectrophotometer UV-2550 Shimadzu, Japan Fluorescence spectrometer F-7000 Hitachi, Japan Nuclear magnetic resonance spectrometer Bruker AV-600 / 500 Bruker (Beijing) Technology Co., Ltd. Microplate reader Bio-rad680 Bio-rad, USA Flow cytometer FACSCalibur BD, USA Inductively coupled plasma mass spectrometry NexIon300X PerkinElmer Laser confocal microscope Zeiss710 Zeiss, Germany Particle size and potential analyzer Zetasizer Nano ZS90 Malvern Instruments Ltd., UK

[0088] Example 1

[0089] Lauric acid (2 g, 10 mmol) and dicyclohexylcarbodiimide (DCC) (1.14 g, 5.53 mmol) were dissolved in 15 mL of dichloromethane. Under nitrogen protection, the mixture was stirred at 25°C for 24 h. The reaction was cooled to room temperature, filtered, and the solid was washed with dichloromethane. The supernatant was taken and dried to obtain lauric anhydride. Lauric anhydride (916.8 mg, 2.4 mmol) and Pt(IV)(OH)2 (200 mg, 0.6 mmol) were weighed and placed in a 50 mL round-bottom flask. 10 mL of anhydrous DMSO and 3 mL of chloroform were added and the mixture was allowed to react at room temperature overnight. The liquid was removed from the 50 mL round-bottom flask and the chloroform was removed using a rotary evaporator. The remaining liquid was transferred to a 50 mL centrifuge tube. 10 times the amount of water was added to precipitate the solution. The solution was then placed in a 4°C refrigerator to cool for half an hour. The solution was centrifuged at 5000 rpm for 5 minutes and the liquid was decanted. The remaining solid was washed three times with water, ether, and acetone to obtain Pt(IV)(C12)2, referred to as Pt-C12. Yield: 75%.

[0090] Weigh Pt-C12 (3.5 mg, 0.005 mmol) and dissolve it in 2 mL of chloroform to obtain a 2.5 mmol / L Pt-C12 chloroform solution. Dissolve m-tetraphenylporphine (TPP) (6.1 mg, 0.01 mmol) in 1 mL of chloroform to obtain a 10 mmol / L TPP chloroform solution. Dissolve all-trans retinoic acid (ATRA) (150 mg, 0.5 mmol) in 1 mL of chloroform to obtain a 500 mmol / L ATRA chloroform solution. Mix 200 μL of the prepared Pt-C12 solution, 200 μL of the TPP solution, and 100 μL of the retinoic acid solution and dilute to 1 mL. Separately, prepare a 0.1 mol / L sodium hydroxide solution for later use.

[0091] Tocopheryl vitamin E polyethylene glycol succinate (TPGS) (7.5 mg) was dissolved in 5 mL of water. The mixture was then shaken with the chloroform solution. 55 μL of 0.1 mol / L NaOH solution was added dropwise. The mixture was sonicated in a 40°C waterbath for 1 hour until the chloroform evaporated and the liquid in the round-bottom flask became clear. This yielded 5 mL of nanomicelles dispersed in water, designated Pt-ATRA-TPP@TPGS. The mixture was centrifuged at 2000 rpm for 5 minutes to remove the precipitate. Inductively coupled plasma mass spectrometry (ICP-MS) analysis revealed a Pt concentration of 99.2 μM, with a Pt loading of 99.2%. UV-visible spectrophotometry determined the TPP concentration to be 296 μM, with a Pt loading of 73.88%. UV-visible spectrophotometry also determined the ATRA content to be approximately 1 mM, with a Pt loading of 10%.

[0092] The performance of the product Pt-ATRA-TPP@TPGS prepared in Example 1 was tested below.

[0093] Test Example 1

[0094] (1) Particle size distribution and stability research

[0095] The particle size distribution of Pt-ATRA-TPP@TPGS was measured using a dynamic light scattering instrument. During the measurement, 20 μL of the prepared nanoparticles were aspirated and diluted into 1 mL of water and 1 mL of DMEM culture medium containing 10% fetal bovine serum, respectively. After being placed at 37°C for a certain period of time (0h, 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h), the particles were measured using a dynamic light scattering instrument to compare the changes in particle size when dispersed in water and DMEM culture medium.

[0096] (2) Particle morphology

[0097] The prepared Pt-ATRA-TPP@TPGS nanoparticles were diluted 10-fold and then dropped onto a 230-mesh copper grid coated with a carbon film using a capillary pipette. Transmission electron microscopy was used to observe the particle morphology. Simultaneously, a pipette was used to drop the liquid onto a silicon wafer for field emission electron microscopy.

[0098] (3) Drug release experiment

[0099] 1 mL of prepared Pt-ATRA-TPP@TPGS nanoparticles was placed in a dialysis bag (1000 molecular weight). The bag was sealed and placed in 10 mL of PBS. The solution was dialyzed for different times (0, 1, 2, 4, 8, 12, 24, and 48 hours). 200 μL of the dialyzed solution was then collected and measured for UV absorbance. After UV absorption measurement, the liquid was recovered and returned to the dialysate.

[0100] (4) Cell culture

[0101] Wash the cells to be treated twice with 2 mL of PBS buffer, add 1 mL of trypsin to digest the cells, and place them in the incubator for about 20 seconds. When the cell morphology changes under the microscope and the cells begin to spherical from the adherent state, quickly remove the trypsin, add 2 mL of serum-containing culture medium, blow the cells on the wall of the culture flask evenly and transfer them to a sterile centrifuge tube, centrifuge at 2000 rpm for 5 minutes, remove the supernatant, add an appropriate amount of culture medium to resuspend, and transfer the cell suspension that meets the experimental requirements to a 25 cm 2 Place the culture bottle in the incubator for further culture or use in various experiments.

[0102] (5) Cytotoxicity test

[0103] A549 and A549R cells were plated at 6×10 3 Cells were inoculated into 96-well plates with a volume of 100 μL per well. The outermost wells were added with the same volume of sterile PBS and cultured in a 37°C, 5% carbon dioxide incubator for 24 h. Different concentrations of drugs were added according to the two-fold dilution method. 3 to 5 parallel groups were set up, and the incubation time was 1 h. After completion, the plates were treated in the dark or illuminated with a 633 nm surface light source (30 mW / cm 2 , 30 min), placed in an incubator and cultured overnight for 24 h, MTT (25 μL / well, 5 mg / mL) was added, incubated for 2-4 h, and then the culture medium was removed. 150 μL of DMSO was added to each well to dissolve the solution, shaken on a shaker, and the absorbance of each well at 490 nm was measured after 15 min. IC 50 The values ​​were calculated using GraphPad Prism software formula (1).

[0104]

[0105] (6) Cellular uptake detection

[0106] Hep-G2 cells were plated at 1×10 6 The cells were seeded into a 6-well plate at a density of 10 μM and cultured in a cell culture incubator for 24 hours. The cells were incubated with 10 μM (Pt concentration) Pt-ATRA-TPP@TPGS for different time periods (0.5 h, 1 h, 2 h, 4 h, 8 h). After incubation, the culture medium was removed and the cells were washed 2 to 3 times with PBS and HBSS. The cells were collected by trypsin digestion and transferred to 500 μL of PBS containing 10% fetal bovine serum. The drug uptake was detected by flow cytometry.

[0107] Under the same culture conditions, cells were pretreated with different endocytosis inhibitors, including chlorpromazine (CPZ, 20 μg / mL), amiloride (AMI, 2 mM), and methyl-β-cyclodextrin (m-β-CD, 5 mM), for 30 minutes. Pt-TPP@TPGS (10 μM) was then added and incubated at 37°C for 4 hours. One group of cells was incubated at 4°C for 4 hours, while a blank group of cells remained untreated and incubated at 37°C for the same period. Following incubation, the culture medium was removed, and the cells were washed two to three times with PBS and HBSS. The cells were harvested by trypsinization and transferred to 500 μL of PBS containing 10% fetal bovine serum. Flow cytometry was used to analyze drug uptake under different inhibitory conditions to determine the uptake pathway.

[0108] Test Example 2

[0109] (1) ROS detection

[0110] Singlet oxygen quantum yield determination

[0111] The sample to be tested was added to the PBS solution of L-Histidine (10 mM) and RNO (20 μM) to make the drug absorbance A 465nm =0.1, through a 465nm surface light source (10mW / cm 2 ) for illumination, and record the absorbance of RNO at 440 nm after every 5 min of illumination to obtain the absorbance change curve under different illumination times.

[0112] Determination of hydroxyl radicals

[0113] The free radicals generated in the sample were measured by monitoring the bleaching amount of methylene blue. The specific detection steps were as follows: TPP and Pt-ATRA-TPP@TPGS solutions (TPP concentration: 10 μM) were mixed with 12.5 mg / mL methylene blue solution, and then the mixture was irradiated with 633 nm light (30 mW / cm 2 ) for different irradiation times (0, 5, 10, 15, 20, 25, 30 min), and the absorbance of methylene blue was measured using a UV-visible spectrophotometer.

[0114] Intracellular ROS detection

[0115] The fluorescence intensity of the reactive oxygen species assay kit DCFH-DA was used to determine the intracellular ROS production. A549 cells were plated at 1×10 4 The cells were seeded into a 48-well plate at a density of 100 μg / cm2 and cultured in a cell culture incubator at 37°C, 5% carbon dioxide, and saturated humidity for 24 h. The cells were incubated with 30 μM TPP and 10 μM Pt-ATRA-TPP@TPGS (TPP content 30 μM) for 1 h. After the drug was removed, the cells were illuminated with a 633 nm surface light source (30 mW / cm2). 2 , 30min), the culture medium was removed after illumination, 20μM DCFH-DA probe was added, and after incubation for 20min, the probe was removed and the fluorescence in the cells was observed under a fluorescence microscope.

[0116] Laser confocal microscopy

[0117] Appropriate Hep-G2 cells were seeded onto confocal laser culture dishes. After adherence, 10 μM of the complex was added. After incubation for 1 hour, the drug was removed and fresh culture medium was added for continued culture. Lyso-Tracker Green (5 μM, 2 hours) and Mito-Tracker Green (150 nM, 30 minutes) were added. After staining, cells were washed two to three times with serum-free culture medium and PBS. The cells were then observed using a Zeiss LSM880 confocal microscope (63× oil immersion lens).

[0118] Live / dead cell staining experiment

[0119] The distribution of cell death was detected by fluorescence intensity using Calcein-AM / PI double staining reagent. 2 x 10 4 A549 cells were seeded in 48-well plates and incubated for 24 h in DMEM containing 10% fetal bovine serum (FBS) for cell adhesion. Then, 3 μM TPP, 1 μM CiPt, 10 μM ATRA, 15 μg / mL TPGS, 1 μM Pt-C12, and 1 μM Pt-ATRA-TPP@TPGS were added to the culture medium, respectively, and incubated for 24 h. AM (4 μM) and PI (6 μM) were added, and the cells were stained for 15 min. The cells were observed under a fluorescence microscope. In addition, TPP and Pt-ATRA-TPP@TPGS were irradiated with a 633 nm light source (30 mW / cm 2 , 30 min) for 1 h, and then incubated in an incubator for 24 h. The cells were then stained and observed.

[0120] Scratch test

[0121] A549 cells were seeded in 6-well plates at a cell density of 1 x 10 5 cells per well and incubated for 24 h in DMEM containing 10% FBS. The central area of cell growth was scratched with a micropipette tip or other hard objects, and the cells in the central area were removed. Then, 3 μM TPP, 1 μM CiPt, 10 μM ATRA, 15 μg / mL TPGS, 1 μM Pt-C12, and 1 μM Pt-ATRA-TPP@TPGS were added to the culture medium, respectively, and the cells were incubated. The cells were then incubated for a certain period of time (1, 2, 3 days), and the 6-well plates were removed. The growth and migration ability of the cells at a specific position were observed and photographed under a microscope. The repair ability of the cells in the scratch area was different between different groups, and the migration and repair abilities of the cells in each group were different.

[0122] Transwell cell migration experiment

[0123] The Transwell chamber is located within a culture plate. The interior of the chamber is called the upper chamber, and the culture plate it is placed in is called the lower chamber. The bottom of the upper chamber is a permeable polycarbonate membrane that separates the culture medium in the upper chamber from the culture medium in the lower chamber. The culture medium in the lower chamber can affect cells in the upper chamber, thereby studying the effect of the fluid in the lower chamber on cell growth and migration. The present invention first prepares a 20% FBS-containing culture medium. This culture medium is then used to prepare 3 μM TPP, 1 μM CiPt, 10 μM ATRA, 15 μg / mL TPGS, 1 μM Pt-C12, and 1 μM Pt-ATRA-TPP@TPGS. 500 μL of each is added to each lower chamber. A 20% FBS-containing culture medium without drugs is also added as a blank control.

[0124] Then use tweezers to place the Transwell chamber into a 24-well plate, trying to avoid bubbles.

[0125] Prepare 3 × 10 4 5 mL of B16F10 cell suspension (500 μL / mL) was added to each upper chamber, and 200 μL was added. The cells were cultured in a cell culture incubator at 37°C, 5% carbon dioxide, and saturated humidity for 24 hours. The Transwell chamber was removed, the culture medium was aspirated, and the cells in the upper chamber were gently wiped with a cotton swab. 600 μL of 4% paraformaldehyde was added to a clean well of a 24-well plate, and the chamber was placed and fixed for 20-30 minutes. Stain with 0.1% crystal violet for 5-10 minutes, wash three times with PBS to remove crystal violet that was not bound to the cells, and gently wipe the upper side of the chamber with a cotton swab to remove the dye that was non-specifically bound to the upper surface of the chamber for subsequent microscopic examination.

[0126] Test Example 3: Tumor Inhibition Experiment

[0127] Establishment of animal model

[0128] A549 cells in the logarithmic growth phase were selected for routine culture. After the cells reached a certain number, they were digested with trypsin for 1 min, and then 2 mL of serum-containing culture medium was added. After being pipetted evenly, the cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 2000 rpm for 5 min. The supernatant was discarded, and the cells were washed 2 to 3 times with PBS, centrifuged, and the supernatant was discarded. Finally, PBS (70%) and high-concentration matrigel (30%) were added to make the cell concentration 1×10 7 / mL and mix thoroughly. Then, aspirate the cell suspension with a syringe, eliminate air bubbles, and inject 100 μL subcutaneously into the right leg of nude mice weighing 20-22 g. Approximately 15 mice were inoculated. Observe the growth of the nude mice and regularly weigh and measure tumor size.

[0129] In vivo tumor inhibition experiment

[0130] The tumor volume grows to 100-150 mm 3 Nude mice were randomly divided into three groups of five: a negative control (saline), a light-exposed group, and a dark-exposed group. The negative control group received an intratumoral injection of 100 μL of saline, while both the light-exposed and dark-exposed groups received 100 μL of 10 μM Pt-ATRA-TPP@TPGS. Eight hours after drug injection, the light-exposed group was irradiated with a 633 nm, 30 mW light source for one hour, while the dark-exposed group remained unexposed. The mice were then observed for 14 days to monitor their growth, and their weight and tumor size were measured regularly.

[0131] 3.3.16.3 Tissue sectioning and analysis

[0132] The tumor inhibition effect of Pt-ATRA-TPP@TPGS and the extent of its side effects on mouse internal organs can be assessed by sectioning tumors and tissues such as the heart, liver, spleen, lung, and kidney. Nude mice were sacrificed, and the tumors, heart, liver, spleen, lung, and kidneys were removed. Each tissue was fixed in 10% paraformaldehyde for 12 hours. After dehydration, transparency, embedding, and sectioning, the tumors, heart, liver, spleen, lung, and kidney tissues were stained with hematoxylin and eosin (HE), and the tumors were also stained with tunnel staining.

[0133] Test Example 4: In vivo anti-metastasis experiment

[0134] Construction of a mouse melanoma lung metastasis model

[0135] Mouse melanoma B16F10 cells in the logarithmic growth phase were selected and cultured in HyClone 1640 medium (containing 10% fetal bovine serum) in a 5% CO2, 37°C cell culture incubator. After the cells grew to a certain number, the medium was removed and the cells were washed twice with PBS. After trypsinization for 1 minute, serum-containing medium was added to terminate the digestion. After pipetting and homogenization, the cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 2000 rpm for 5 minutes. The supernatant was discarded and the cells were washed 2 to 3 times with PBS. The cells were centrifuged and the supernatant was discarded. Finally, PBS was added to make the cell concentration 2×10 7 Aspirate the cell suspension with a syringe, eliminate air bubbles, and inject 100 μL of the cell suspension into the tail vein of 21 6-8 week old C57BL / 6J male mice.

[0136] In vivo anti-metastasis experiment

[0137] Mice injected with a B16F10 cell suspension were treated with 30 μM TPP, 10 μM CiPt, 100 μM ATRA, 150 μg / mL TPGS, 10 μM Pt-C12, and 10 μM Pt-ATRA-TPP@TPGS, respectively, in three parallel experiments. Another group received saline alone. After 21 days of observation, the mice were dissected, their lungs removed, sectioned, and photographed to observe the presence of melanoma in the lungs. The lungs were also stained with H&E.

[0138] Test results

[0139] (1) Structural characterization of the complex

[0140] In an aqueous solution, when the surfactant reaches a certain concentration, the molecules self-assemble to form orderly arranged micelles. The mechanism of micelle formation is that after the surfactant with adsorption capacity reaches a saturated state, the excess surfactant is dispersed in the aqueous solution. Due to the presence of its hydrophobic groups, the repulsive force between water molecules and the surfactant is stronger than the attractive force. The hydrophobic groups associate under the action of van der Waals forces to form the micelle core, and the hydrophilic groups form the micelle outer layer outward, which is stably dispersed in the aqueous solution. However, cisplatin is soluble in water and is not easily carried by nanomicelles. Therefore, the embodiment of the present invention modifies cisplatin by bonding lauric anhydride at both ends to make it less soluble in water. The nuclear magnetic resonance characterization of the synthesized Pt-C12 of the present invention is as follows Figure 1 As shown. 1HNMR shows that the peaks of Pt-C12 prepared by the present invention have clear attribution. Before bonding with lauric anhydride, the amino group of the platinum ligand is at 5.5ppm, but after bonding, its peak changes to 6.5ppm. The methylene peak (-CH2-CH2-) on lauric anhydride appears at 1.25ppm, and the ratio corresponds to that in the structural formula, proving that the present invention successfully synthesized Pt-C12.

[0141] (2) Characterization of Pt-ATRA-TPP@TPGS nanomaterials

[0142] The nanoparticles prepared in the present invention were characterized by transmission electron microscopy and field emission electron microscopy. Figure 2 and A and B, where Figure 2 Figure A is a SEM image of Pt-ATRA-TPP@TPGS, and Figure B is a TEM image of Pt-ATRA-TPP@TPGS. Transmission electron microscopy results show that the synthesized Pt-ATRA-TPP@TPGS particles are relatively uniform in size, with a particle size of approximately 60 nm, which is suitable for use as anti-metastasis nanomedicines.

[0143] The UV absorption peaks of TPP, ATRA and Pt-ATRA-TPP@TPGS are as follows: Figure 3As shown in A in the figure. Before and after the addition of ATRA synthesized nanoparticles, the ultraviolet absorption of TPP is roughly the same, with no red shift or blue shift. At the same time, because the ultraviolet has absorption at 643nm, this also makes it possible to use red light for cell experiments and in vivo experiments in the present invention. The drug release curve over time is shown in Figure 2. Figure 3 As shown in Figure 5B, the drug release increases with time.

[0144] The particle size distribution of Pt-ATRA-TPP@TPGS is as follows Figure 3 As shown in Figure C, 97% of the nanoparticles have a particle size distribution range of 43 to 170 nm, with the highest peak being 60 nm. Figure 3 As shown in D in the figure, the ζ potential decreases with the increase of pH value. When pH = 4, ζ = 2.653; when pH = 6.8, ζ = -1.119; when pH = 7.2, ζ = -1.505; when pH = 9, ζ = -4.138.

[0145] (3) Production of ROS

[0146] Based on dimethyl-4-nitrosoaniline (N,N-Dimethyl-p-nitrosoaniline; RNO) as a selective receptor for singlet oxygen, it produces a decolorization effect in the presence of singlet oxygen, showing a decrease in the absorbance peak under a UV-visible spectrophotometer (440nm wavelength). Figure 4 As shown, Figure 4 For RNO / His reagent detection and different drugs 633nm (30mW / cm 3 ) produced under light 1 O2, Figure 4 The A in it is TPP dark; Figure 4 B in the figure is TPP illumination; Figure 4 C in the figure is Pt-ATRA-TPP@TPGS dark; Figure 4 D in the equation is the illumination of Pt-ATRA-TPP@TPGS. Figure 4 It can be seen that under dark conditions, neither TPP nor Pt-ATRA-TPP@TPGS produced singlet oxygen. However, under light conditions, both TPP and Pt-ATRA-TPP@TPGS bleached RNO, and the bleaching effect became more pronounced with longer light exposure. This indicates that replacing oleic acid with ATRA did not affect the photodynamic efficiency of the particles.

[0147] In addition to RNO, methylene blue bleaching method is also a common method for detecting hydroxyl radicals. Figure 4 As shown, Figure 5 Methylene blue bleaching method for detecting hydroxyl radicals, Figure 5The A in it is TPP dark; Figure 5 B in the figure is TPP illumination; Figure 5 C in the figure is Pt-ATRA-TPP@TPGS dark; Figure 5 D in the equation is the illumination of Pt-ATRA-TPP@TPGS. Figure 5 It can be seen that under dark conditions, methylene blue is not bleached, but under 633nm light irradiation, methylene blue is bleached more and more with increasing illumination time. This shows that under light conditions, both TPP and Pt-ATRA-TPP@TPGS can generate hydroxyl radicals, and the addition of ATRA has no negative impact on PDT.

[0148] In order to verify whether TPP and Pt-ATRA-TPP@TPGS have the same effect after entering cells and produce singlet oxygen, the present invention used DCFH-DA reactive oxygen species detection kit to test, such as Figure 5 As shown, Figure 6 The results of singlet oxygen detection in cells show that under dark conditions, both TPP and Pt-ATRA-TPP@TPGS have no fluorescence, indicating that neither of them produces singlet oxygen under dark conditions. However, under light conditions, they both emit green fluorescence, indicating that both TPP and Pt-ATRA-TPP@TPGS have entered the cells and produced singlet oxygen under light. Figure 6 As shown in the figure, at the same concentration, the singlet oxygen fluorescence intensity generated after Pt-ATRA-TPP@TPGS enters the cell is greater than the fluorescence of TPP, which also shows that after being made into nanoparticles, the uptake efficiency of TPP is higher than that in the free state.

[0149] (4) Cytotoxicity

[0150] In order to understand the toxicity of each drug to cells, the present invention uses the MTT method to detect cytotoxicity. Figure 6 The cytotoxicity of the drugs to A549 and A549R cells was evaluated by MTT assay. Figure 7 As shown in Figure 2, under 633 nm light conditions, the toxicity of TPP and Pt-ATRA-TPP@TPGS to cells was significantly greater than that under dark conditions, proving that TPP and Pt-TPP@TPGS have the effect of photodynamic therapy. Figure 7 A) and A549R( Figure 7 Compared with the cells in B), Pt-ATRA-TPP@TPGS has better cytotoxicity than cisplatin. The IC 50 The values ​​are shown in Table 3.

[0151] Table 3 IC of each drug 50 value

[0152]

[0153] In order to observe the cytotoxicity more intuitively, the present invention adopts CalceinAM / PI live and dead cell staining kit to detect cells. Because dead cells lack esterase or esterase activity is very low, the living cells containing esterase after CalceinAM enters the cells can produce Calcein, while dead cells cannot or rarely can produce Calcein, so only living cells will be dyed as strong green fluorescence, and dead cells cannot be dyed or dye very weakly. Nucleic acid red fluorescent dye propidium iodide (PropidiumIodide, PI) can only dye dead cells whose cell membrane integrity is destroyed because it cannot penetrate the cell membrane of living cells. Therefore, CalceinAM is used in combination with propidium iodide to carry out dual fluorescent staining to living cells and dead cells simultaneously, which can be used for the detection of cell activity and cytotoxicity.

[0154] like Figure 7 As shown, Figure 8 Live and dead cells were stained under dark conditions under treatment with various drugs. The cytotoxicity of Pt-ATR-TPP@TPGS at the same concentration was much greater than that of other drugs.

[0155] At the same time, if Figure 8 As shown, Figure 9 The live and dead cell staining of TPP and Pt-ATRA-TPP@TPGS under light and dark conditions showed that the cytotoxicity of TPP or Pt-ATR-TPP@TPGS under light was greater than that under dark conditions at the same concentration, which proved the photodynamic therapy effect of TPP and Pt-ATR-TPP@TPGS.

[0156] (5) Anti-metastasis effect

[0157] To verify the anti-metastatic effect of Pt-ATR-TPP@TPGS, the researchers conducted a scratch assay. The scratch assay is a simple, rapid, and convenient in vitro method for studying cell migration. The principle is that when cells proliferate to a confluent monolayer, a blank area, called a "scratch," is artificially created in the confluent monolayer. Cells on either side of the scratch gradually migrate into the blank area, healing the scratch. This assay, to some extent, mimics the lateral cell migration process in vivo.

[0158] like Figure 9 As shown, compared with the blank group and other drugs, the width of the scratches remained basically unchanged in the Pt-ATR-TPP@TPGS environment, proving that Pt-ATR-TPP@TPGS can effectively inhibit cancer cell migration, and nanomicelle-encapsulated ATRA has higher anti-metastasis ability than free ATRA.

[0159] Transwell technology, as an experimental technique, primarily involves the Transwell chamber, which is placed within a culture plate. The inner chamber is called the upper chamber, while the inner chamber is called the lower chamber. Each chamber contains culture medium and is separated by a polycarbonate membrane. Cells are seeded in the upper chamber. Because the polycarbonate membrane is permeable, components in the culture medium in the lower chamber can affect cells in the upper chamber, allowing the effects of the culture medium in the lower chamber on cell growth, proliferation, motility, and migration to be studied. Using polycarbonate membranes with varying pore sizes and treatments, a wide range of studies can be conducted, including cell co-culture, cell chemotaxis, cell migration, and invasion.

[0160] like Figure 10 As can be seen, virtually no cells passed through the polycarbonate membrane due to the Pt-ATR-TPP@TPGS, demonstrating that Pt-ATR-TPP@TPGS can successfully inhibit cell migration. The migration inhibition abilities of other components within the nanomicelles are shown in the figure.

[0161] A melanoma lung metastasis model was established in C57BL / 6J mice to lay the experimental foundation for subsequent studies on B16F10 anti-metastasis experiments. Figure 11 and 13 As shown, Figure 12 A mouse melanoma lung metastasis model. Figure 12 H&E staining of the lungs in a mouse melanoma lung metastasis model. During implantation of the B16F10 metastatic cell line, nanomicelle-encapsulated ATRA exhibited higher anti-metastatic efficacy than free ATRA. Similar anti-metastatic effects were also observed in the lungs.

[0162] (6) Tumor inhibition experiment

[0163] In order to prove that the prepared anti-metastatic nanomedicine has a good effect at the animal level, the present invention selected the A549 subcutaneous tumor model to verify the tumor inhibition effect of Pt-ATRA-TPP@TPGS. The components set were: saline group, light group and dark group, in order to verify the photodynamic therapy effect of TPP material. Figure 13 shown. Figure 14 A in the figure represents the change of tumor volume over time; Figure 14 B in the figure represents the change of relative body weight of mice over time; Figure 14 C in the figure shows the tumor status of mice after treatment; Figure 14 D in the equation is the tumor mass of the mouse; Figure 14 It can be seen that the tumor volume of the saline group remained at 400 mm in the first five days after administration. 3Within five days, however, the tumors grew rapidly and became uncontrollable. The tumor volume of mice treated with Pt-ATRA-TPP@TPGS was significantly reduced compared to the saline group, regardless of whether they were exposed to light or darkness. The light group exhibited a more effective tumor suppression effect than the dark group, with tumors in this group showing a decreasing trend.

[0164] In order to further verify the toxic and side effects of the anti-metastatic nanomedicine, the present invention performed H&E staining on the heart, liver, spleen, lung and kidney of mice. The results are as follows Figure 15 As shown, while killing tumor cells, it basically did not damage the heart, liver, spleen, lung and kidney tissue structures of mice, showing good anti-cancer activity and very few toxic side effects. Figure 16 The TUNEL staining results of the tumor. Figure 16 As shown, blue stains the cell nucleus and yellow indicates apoptosis. Figure 16 As can be seen from the results, the saline group had no effect on the tumor, while the tumor tissues of the treatment groups suffered a certain degree of damage, especially the light-irradiated group. This indicates that light irradiation enhances the tumor-suppressing effect of the nano-micelles.

[0165] The anti-metastatic nanodrug, Pt-ATRA-TPP@TPGS, uses TPGS nanomicelles as a carrier to co-deliver Pt-C12, TPP, and ATRA. In addition to exhibiting excellent chemotherapy and photodynamic therapy effects, it can also be used for anti-metastatic tumor treatment. The drug disperses evenly in water, exhibits excellent stability, and exhibits strong cytotoxicity, demonstrating superior anti-metastatic efficacy. Pt-ATRA-TPP@TPGS exhibits superior anti-metastatic activity compared to free ATRA.

[0166] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. An anti-tumor metastasis nanomedicine, characterized in that: The invention comprises vitamin E polyethylene glycol succinate nanomicelles, and a lauric acid-modified tetravalent platinum prodrug, all-trans retinoic acid, and a hydrophobic photosensitizer encapsulated in the vitamin E polyethylene glycol succinate nanomicelles; the lauric acid-modified tetravalent platinum prodrug is obtained by reacting Pt(NH3)2(OH)2Cl2 and lauric anhydride, the hydrophobic photosensitizer is selected from m-tetraphenylporphine and / or hematophyll methyl ether, and the chemical structure of the lauric acid-modified tetravalent platinum prodrug is shown in Formula 2: Formula 2.

2. The anti-tumor metastasis nanomedicine according to claim 1, characterized in that The particle size of the anti-tumor metastasis nanomedicine is 60 nm.

3. The anti-tumor metastasis nanomedicine according to claim 1, characterized in that The preparation method of the lauric acid-modified tetravalent platinum prodrug comprises the following steps: Mixing lauric anhydride, a complex Pt(NH3)2(OH)2Cl2, a first organic solvent, and a second organic solvent for a grafting reaction to obtain a grafting reaction liquid; the first organic solvent is an organic solvent miscible with water, and the second organic solvent is an organic solvent immiscible with water; The grafting reaction solution is removed from the second organic solvent and then mixed with water, and the lauric acid-modified tetravalent platinum prodrug is obtained after solid-liquid separation.

4. The method for preparing the anti-tumor metastasis nanomedicine according to any one of claims 1 to 3, characterized in that: The following steps are involved: mixing a lauric acid-modified tetravalent platinum prodrug, all-trans retinoic acid, a hydrophobic photosensitizer, and a third organic solvent to obtain a drug solution; dissolving vitamin E polyethylene glycol succinate in water to obtain a vitamin E polyethylene glycol succinate solution; mixing the drug solution and the vitamin E polyethylene glycol succinate solution to obtain a mixed liquid; The mixed liquid is added dropwise to an inorganic strong alkali aqueous solution for mixing, and then the third organic solvent is removed to obtain a dispersion of the anti-tumor metastasis nanomedicine.

5. The preparation method according to claim 4, characterized in that The molar ratio of the lauric acid-modified tetravalent platinum prodrug to the hydrophobic photosensitizer is 0.0025:0.

01.

6. The preparation method according to claim 4 or 5, characterized in that The molar ratio of the lauric acid-modified tetravalent platinum prodrug to all-trans retinoic acid is 0.005:0.

5.

7. The preparation method according to claim 4 or 5, characterized in that The mass ratio of the lauric acid-modified tetravalent platinum prodrug to vitamin E polyethylene glycol succinate is 3.5:7.

5.

8. Use of the anti-tumor metastasis nanomedicine according to any one of claims 1 to 3 or the anti-tumor metastasis nanomedicine prepared by the preparation method according to any one of claims 4 to 7 in the preparation of anticancer drugs.

9. A chemotherapy-photodynamic therapy combined with anti-tumor metastasis diagnostic and therapeutic reagent, characterized in that: The invention comprises the anti-tumor metastasis nanomedicine according to any one of claims 1 to 3 or the anti-tumor metastasis nanomedicine prepared by the preparation method according to any one of claims 4 to 7.

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