Anti-metastasis nano-drug, preparation method and application thereof, and chemotherapy-photodynamic combined anti-metastasis diagnosis and treatment reagent

Abstract

The invention belongs to the technical field of anti-cancer drugs, and particularly relates to an anti-metastasis nano-drug, a preparation method and application thereof, and a chemotherapy-photodynamic combined anti-metastasis diagnosis and treatment reagent. The anti-metastasis nano-drug provided by the invention comprises a vitamin E polyethylene glycol succinate nano-micelle, and a lauric acid modified tetravalent platinum prodrug, all-trans retinoic acid and a hydrophobic photosensitizer which are coated in the vitamin E polyethylene glycol succinate nano-micelle. After the anti-metastasis nano-drug provided by the invention is passively transported into tumor cells in a targeted manner through an EPR effect, the hydrophobic photosensitizer can increase the generation of singlet oxygen under the irradiation of red light and cooperates with a chemotherapy drug (tetravalent platinum) to act together, chemotherapy and photodynamic therapy are combined, drug resistance can be overcome, and a good treatment effect is achieved; meanwhile, the anti-metastasis nano-drug provided by the invention has relatively strong cytotoxicity, shows an 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 diagnosis and treatment 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. At present, more than 500 related anti-tumor metastasis agents have been studied and reported. The most studied anti-tumor metastasis drugs mainly include angiogenesis inhibitors and matrix metalloproteinase inhibitors. Anti-tumor metastasis drugs approved for clinical use by the US Food and Drug Administration (FDA) also belong to these two categories. However, these anti-tumor metastasis drugs have almost no effect on patients with advanced disease, especially due to their non-selective molecular inhibition and no significant improvement in patient survival. As a result, the anti-tumor metastasis effect of the currently used anti-tumor metastasis drugs is weak.

[0003] All-trans retinoic acid (ATRA) is a metabolite of vitamin A acid in the body. As the first choice for leukemia treatment, it has been widely studied due to its therapeutic efficiency in other solid cancers. It can promote cell differentiation, has low toxicity, and can regulate the plasticity and motility of cancer cells. It has the effect of inhibiting tumor metastasis and is widely used in the treatment of cancers such as squamous cell carcinoma and melanoma. However, due to its short half-life, poor water solubility, and easy inactivation by light, oxygen, and heat, its efficacy in treating solid tumors is poor.

[0004] At present, nanocarriers are often used to increase their water solubility and stability, significantly increasing their in vivo circulation time. In addition, due to the high permeability and retention effect (EPR effect) of solid tumors, anti-metastatic nanomedicines can be passively targeted and enriched in tumor tissues. So far, there have been reports on the use of liposomes, polymers, etc. to load ATRA for cancer treatment. However, the therapeutic effect of anti-tumor metastasis is still poor. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-metastatic nano drug and its preparation method and application, and a chemotherapy-photodynamic combined anti-metastatic diagnostic and therapeutic reagent. The anti-metastatic nano drug 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 invention provides an anti-metastasis nano drug, comprising vitamin E polyethylene glycol succinate nano micelles, and lauric acid-modified tetravalent platinum prodrug, all-trans retinoic acid and a hydrophobic photosensitizer encapsulated in the vitamin E polyethylene glycol succinate nano micelles; 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 method for preparing 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 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 nano drug 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 for mixing, and then the third organic solvent is removed to obtain the anti-transfer 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 nano drug described in the above technical solution or the anti-metastasis nano drug 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 nano drug described in the above technical solution or the anti-metastasis nano drug prepared by the preparation method described in the above technical solution.

[0023] The invention provides an anti-metastasis nano drug, comprising vitamin E polyethylene glycol succinate nano micelles, and lauric acid-modified tetravalent platinum prodrug, all-trans retinoic acid (ATRA) and a hydrophobic photosensitizer encapsulated in the vitamin E polyethylene glycol succinate nano micelles; the lauric acid-modified tetravalent platinum prodrug is obtained by reacting Pt(IV)(OH)2 and lauric anhydride. The invention uses a kinetically stable core-shell structure nano-micelle 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, and has good encapsulation and high drug loading, thereby realizing the co-transportation of cisplatin prodrug, ATRA and photosensitizer; after the anti-metastatic nano-drug provided by the 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 cooperates with a chemotherapeutic drug (tetravalent platinum) to combine chemotherapy with photodynamic therapy, thereby overcoming drug resistance and achieving a good therapeutic effect; at the same time, the anti-metastatic nano-drug provided by the invention has strong cytotoxicity, shows an excellent anti-metastatic effect, 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 comprises the following steps: lauric anhydride, complex [Pt(NH2)2(OH)2Cl2], a first organic solvent and a second organic solvent are mixed 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; the grafting reaction liquid 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. In the present invention, the cisplatin prodrug is easily soluble in water, and thus is not easily supported by the vitamin E polyethylene glycol succinate nano-micelles. The present invention modifies the complex [Pt(NH2)2(OH)2Cl2] by lauric anhydride, chemically bonds lauric anhydride to the complex [Pt(NH2)2(OH)2Cl2] molecule, and enhances the hydrophobicity of cisplatin prodrug. The obtained lauric acid-modified tetravalent platinum prodrug can be effectively coated by nano micelles, and has good encapsulation and high drug loading. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 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 distribution of TPP, ATRA, and Pt-ATRA-TPP@TPGS in Example 1, and the zeta potential of Pt-ATRA-TPP@TPGS at different pH values;

[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 it is Pt-ATRA-TPP@TPGS illumination;

[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 it is Pt-ATRA-TPP@TPGS illumination;

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

[0031] Figure 7 The results of cytotoxicity test 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] Fig. 9 Live and dead cell staining results of TPP and Pt-ATRA-TPP@TPGS under light and dark conditions;

[0034] Fig.10 is the scratch test result;

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

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

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

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

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

[0040] Fig.16 This is the TUNEL staining result of the tumor;

[0041] Fig.17 The synthetic route of lauric acid-modified tetravalent platinum prodrug Pt-C12. DETAILED DESCRIPTION

[0042] The invention provides an anti-metastasis nano drug, comprising vitamin E polyethylene glycol succinate nano micelles, and lauric acid-modified tetravalent platinum prodrug, all-trans retinoic acid and a hydrophobic photosensitizer encapsulated in the vitamin E polyethylene glycol succinate nano micelles; 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 structural formula of the vitamin E polyethylene glycol succinate provided by the present invention is shown in Formula 1:

[0046]

[0047] The 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 (diameter 12-15 nm) and exhibit the properties of a solubilizer, emulsifier, dispersant and gelling agent. Its aggregation behavior can be regulated together 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 TPGS with adsorption capacity 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 force to form the inner core of the micelle, and the hydrophilic groups face outward to form the outer layer of the micelle, which is stably dispersed in the aqueous solution.

[0049] The anti-metastasis nano drug provided by the present invention comprises a lauric acid-modified tetravalent platinum prodrug encapsulated in the vitamin E polyethylene glycol succinate nano micelle. 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 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 preparation method of lauric anhydride preferably comprises the following steps: in a protective gas atmosphere, lauric acid, a dehydrating agent and an organic solvent are mixed for dehydration reaction to obtain 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 has no special requirements for the amount of the organic solvent, as long as the dehydration reaction is carried out smoothly. In the present invention, the protective gas is preferably N2. The temperature of the dehydration reaction is preferably 25°C, and the time is preferably 24h; the dehydration reaction is carried out under stirring. After the dehydration reaction is completed, a dehydration reaction liquid is obtained. The present invention preferably separates the dehydration reaction liquid cooled to room temperature into solid and liquid, and the obtained solid product is washed to obtain a washing supernatant; the washing supernatant is dried to obtain lauric anhydride. In the present invention, the solid-liquid separation is preferably suction filtration, the reagent used for washing is preferably dichloromethane; and the drying is preferably 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 is carried out smoothly.

[0057] In the present invention, the grafting reaction is preferably carried out at room temperature. After the grafting reaction is completed, the present invention preferably mixes the grafting reaction solution with water, and then cools it at 0-4°C, separates the obtained cooled solution into solid and liquid, and washes the obtained solid product 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 centrifugation, the speed of the centrifug is preferably 5000rpm, and the time is preferably 5min. The washing is preferably: washing with water, ether and acetone in sequence, and washing each solvent 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 invention comprises a hydrophobic photosensitizer encapsulated in the vitamin E polyethylene glycol succinate nano micelle.

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

[0062] In the present invention, the chemical structural formula 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 micelle.

[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 nano drug is preferably 60 nm.

[0067] In the anti-metastasis nano drug 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 nano drug 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 for mixing, and then the third organic solvent is removed to obtain the anti-transfer nano drug dispersion.

[0073] The present invention mixes a lauric acid-modified tetravalent platinum prodrug, all-trans retinoic acid, a hydrophobic photosensitizer and a third organic solvent to obtain 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; 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 2.5mmol / L; the molar concentration of the photosensitizer solution is preferably 10mmol / L; the molar concentration of the all-trans retinoic acid solution is preferably 500mmol / 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 drops the mixed liquid into an inorganic strong alkali aqueous solution for mixing, and then removes the third organic solvent to obtain a dispersion of the anti-metastasis nanomedicine. 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 to 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, the evaporation is carried out under ultrasonic oscillation, the evaporation temperature is preferably 40°C, the evaporation is carried out under water bath conditions, and the ultrasonic oscillation time is preferably 1h, until the third organic solvent is volatilized.

[0078] The anti-metastasis nano drug dispersion provided by the present invention is preferably an anti-metastasis nano drug aqueous dispersion. In the present invention, in the anti-metastasis nano drug aqueous dispersion: the Pt concentration is 99.2 μM, the loading rate is as high as 99.2%, and the TPP content is measured by UV-visible spectrophotometer, and the TPP concentration is 296 μM, and the loading rate is 73.88%. The ATRA content is measured by UV-visible spectrophotometer, and the ATRA is about 1 mM, and the loading rate is 10%.

[0079] The present invention provides the use of the anti-metastasis nano drug described in the above technical solution or the anti-metastasis nano drug prepared by the preparation method described in the above technical solution in the preparation of anticancer drugs.

[0080] The present invention provides a chemotherapy-photodynamic combined anti-metastasis diagnostic and therapeutic reagent, which includes the anti-metastasis nano drug described in the above technical solution or the anti-metastasis nano drug prepared by the preparation method described in the above technical solution.

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

[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 High-speed refrigerated centrifuge 5418R Eppendorf, Germany Ultra-clean sterile workbench SW-CJ-1FD Sujing Antai Carbon dioxide incubator HF90 Likang Biotechnology UV spectrophotometer UV-2550 Shimadzu Fluorescence Spectrometer F-7000 Hitachi, Japan Nuclear Magnetic Resonance Spectrometer BrukerAV-600 / 500 Bruker (Beijing) Technology Co., Ltd. ELISA reader Bio-rad680 American Bole Flow Cytometer FACSCalibur American BD Inductively Coupled Plasma Mass Spectrometry NexIon300X PerkinElmer Laser confocal microscopy Zeiss710 Zeiss, Germany Particle size and potential analyzer ZetasizerNanoZS90 Malvern Instruments Ltd.

[0088] Example 1

[0089] Weigh lauric acid (2g, 10mmol), dicyclohexylcarbodiimide (Dicyclohexylcarbodiimide) DCC (1.14g, 5.53mmol) and dissolve in 15mL dichloromethane. Under N2 protection, stir and react at 25℃ for 24h. Cool the reaction to room temperature, filter, wash the solid with dichloromethane, take the supernatant, spin dry to obtain lauric anhydride. Weigh lauric anhydride (916.8mg, 2.4mmol) and Pt(Ⅳ)(OH)2 (200mg, 0.6mmol) in a 50mL round-bottom flask, add 10mL anhydrous DMSO and 3mL chloroform, and react overnight at room temperature. Take out the liquid in a 50mL round-bottom flask, spin out the chloroform with a rotary evaporator, transfer the remaining liquid into a 50mL centrifuge tube, add 10 times water, precipitate, and then put it in a 4℃ refrigerator to cool for half an hour, centrifuge at 5000rpm for 5min, and pour out the liquid. The remaining solid was washed three times with water, ether and acetone respectively 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 chloroform solution of Pt-C12, 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, take 200 μL of the above-prepared Pt-C12 solution, 200 μL of TPP solution, and 100 μL of retinoic acid solution, mix them together, and dilute to 1 mL. In addition, prepare a 0.1 mol / L sodium hydroxide solution for use.

[0091] Tocopherol vitamin E polyethylene glycol succinate (TPGS) (7.5 mg) was dissolved in 5 mL of water, and the two phases were mixed with the above-mentioned chloroform solution and shaken, and 55 μL of 0.1 mol / L NaOH solution was added dropwise, and ultrasonic vibration was performed in a 40°C water bath for 1 hour until the chloroform evaporated and the liquid in the round-bottom flask became clear, and 5 mL of nano-micelles dispersed in water were obtained, named Pt-ATRA-TPP@TPGS. Centrifuged at 2000 rpm for 5 minutes to remove the precipitate. After determination by inductively coupled plasma mass spectrometry (ICP-MS), it was calculated that the Pt concentration was 99.2 μM, and the loading rate was as high as 99.2%. The TPP content was determined by UV-visible spectrophotometer, and the TPP concentration was 296 μM, and the loading rate was 73.88%. The ATRA content was measured by UV-visible spectrophotometer, and it was found that ATRA was about 1 mM and the loading rate was 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 study

[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), they were measured using a dynamic light scattering instrument to compare the changes in particle size when the particles were dispersed in water and DMEM culture medium.

[0096] (2) Particle morphology

[0097] The prepared Pt-ATRA-TPP@TPGS nanoparticles were diluted 10 times and then dropped onto a 230-mesh copper grid containing a carbon film using a capillary tube. The particle morphology was observed using a transmission electron microscope. At the same time, the liquid was dropped onto a silicon wafer using a pipette and the particle morphology was observed using a field emission electron microscope.

[0098] (3) Drug release experiment

[0099] 1 mL of prepared Pt-ATRA-TPP@TPGS nanoparticles was taken into a dialysis bag (1000 molecular weight), the dialysis bag was sealed and placed in 10 mL of PBS, and dialyzed for different time periods (0, 1, 2, 4, 8, 12, 24, 48 h), and then 200 μL of the dialyzed solution was taken to measure the UV absorption. After measuring the UV absorption, the liquid was recovered in the dialysate.

[0100] (4) Cell culture

[0101] After washing the cells to be treated twice with 2 mL 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 become 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 bottle 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 for various experiments.

[0102] (5) Cytotoxicity test

[0103] A549 and A549R cells were plated at 6×10 3 The cells were inoculated into a 96-well plate, 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 , 30min), put it in the incubator and culture it overnight for 24h, then add MTT (25μL / well, 5mg / mL), incubate it for 2-4h, remove the culture medium, add 150μL DMSO to each well to dissolve it, shake it on a shaker, and detect the absorbance of each well at 490nm after 15min. IC 50 The values ​​were calculated using the GraphPad Prism software formula (1).

[0104]

[0105] (6) Cellular uptake detection

[0106] Hep-G2 cells were plated at 1×10 6 The cells were inoculated into a 6-well plate at a density of 1 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 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, and Pt-TPP@TPGS (10 μM) was added and incubated at 37°C for 4 hours. One group of cells was incubated at 4°C for 4 hours, and the cells in the blank group were not treated and incubated at 37°C for the same time. After incubation, the culture medium was removed, and the cells were washed 2 to 3 times with PBS and HBSS, and the cells were collected by trypsin digestion, and the cells were transferred to 500 μL PBS containing 10% fetal bovine serum. The drug uptake under different inhibitory conditions was detected by flow cytometry to determine the uptake pathway.

[0108] Test Example 2

[0109] (1) ROS detection

[0110] Singlet oxygen quantum yield determination

[0111] Add the sample to be tested 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 minutes 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 illuminated 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 kit DCFH-DA was used to determine the intracellular ROS production. A549 cells were plated at 1×10 4 The cells were inoculated into a 48-well plate at a density of 1.50 μg / cm2 and cultured in a cell culture incubator at 37°C, 5% carbon dioxide and saturated humidity for 24 h with culture medium containing 10% fetal bovine serum. The cells were incubated with 30 μM TPP and 10 μM Pt-ATRA-TPP@TPGS (TPP content 30 μM) for 1 h, and then illuminated with a 633 nm surface light source (30 mW / cm2) after the drug was removed. 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 inoculated into laser confocal culture dishes, and 10μM complex was added after adherent growth. After incubation for 1h, the drug was removed and new culture medium was added to continue culture. Lyso-Tracker Green (5μM, 2h) and Mito-Tracker Green (150nM, 30min) were added. After staining, the cells were washed 2-3 times with serum-free culture medium and PBS, and observed with a Zeiss LSM880 confocal microscope (63× oil lens).

[0118] Live / dead cell staining assay

[0119] The fluorescence intensity of Calcei-AM / PI double staining reagent was used to detect the distribution of cell death. 4 A549 cells were inoculated into 48-well plates and grown in 10% fetal bovine serum for 24 h. 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 and incubated for 24 h. AM (4 μM) and PI (6 μM) were added and stained for 15 min. The cells were then examined under a fluorescence microscope. In addition, TPP and Pt-ATRA-TPP@TPGS were illuminated with a 633 nm surface light source (30 mW / cm 2 , 30min), put it in the incubator for 24h, and then stain it for observation.

[0120] Scratch test

[0121] A549 cells were plated at 1×10 5 The cells were inoculated into a 6-well plate at a density of 100 μM and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) for 24 h. The central area of ​​cell growth was scratched with a micropipette tip or other hard objects, and the cells in the central part were removed. The culture medium containing 3 μM TPP, 1 μM CiPt, 10 μM ATRA, 15 μg / mL TPGS, 1 μM Pt-C12, and 1 μM Pt-ATRA-TPP@TPGS was added and incubated. Then the cells were cultured for the corresponding time (1, 2, and 3 days), and the six-well plate was taken out. The growth and migration ability of cells in specific positions were observed and photographed under a microscope. The difference in the repair ability of cells in different groups for the scratch area can be used to determine the difference in the migration and repair ability of cells in each group.

[0122] Transwell cell migration assay

[0123] The Transwell chamber is in a culture plate, the chamber is called the upper chamber, the culture plate in which the chamber is placed is called the lower chamber, the bottom of the upper chamber is a layer of polycarbonate membrane, this membrane is permeable, and separates the culture solution in the upper chamber from the culture solution in the lower chamber, and the culture solution in the lower chamber can affect the cells in the upper chamber, so as to study the effect of the liquid in the lower chamber on the growth and migration of cells. The present invention first prepares a 20% FBS-containing culture medium, and uses the culture medium to prepare 3μM TPP, 1μM CiPt, 10μATRA, 15μg / mL TPGS, 1μMPt-C12, and 1μM Pt-ATRA-TPP@TPGS, and adds 500μL to each lower chamber, and adds a 20% FBS-containing culture medium without drugs 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 / mL B16F10 cell suspension 5mL, add 200μL to each upper chamber, place in a cell culture incubator at 37℃, 5% carbon dioxide, saturated humidity for 24 hours, remove the Transwell chamber, aspirate the culture medium, and gently wipe the cells in the upper chamber with a cotton swab. Add 600μL of 4% paraformaldehyde to a clean well of a 24-well plate, put the chamber in and fix for 20-30min. Stain with 0.1% crystal violet for 5-10min, wash 3 times with PBS to remove the crystal violet that is not bound to the cells, and gently wipe the upper side of the chamber with a cotton swab to wipe off the dye that is 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 grew to a certain number, they were digested with trypsin for 1 min, and then 2 mL of serum-containing culture medium was added. After being blown 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 PBS was added for washing 2 to 3 times. The supernatant was discarded, and PBS (70%) and high-concentration matrix gel (30%) were finally added to make the cell concentration 1×10 7 / mL, mix well. Then use a syringe to draw up the cell suspension, expel air bubbles, and inject 100μL subcutaneously into the right leg of nude mice weighing 20-22g. About 15 mice were inoculated in total. The growth of nude mice was observed, and the nude mice were weighed regularly and the tumor size was measured.

[0129] In vivo tumor inhibition experiment

[0130] The tumor volume grows to 100-150 mm 3 The nude mice were randomly divided into three groups, 5 in each group, namely the negative control (normal saline) group, the light group and the dark group. The negative control group was injected with 100μL normal saline intratumorally, and the light group and the dark group were injected with 100μL 10μM Pt-ATRA-TPP@TPGS. 8h after drug injection, the light group mice were irradiated with 633nm, 30mW light source for 1h, and the dark group did not need light. The nude mice were observed for 14 days to observe their growth, and their weight and tumor size were measured regularly.

[0131] 3.3.16.3 Tissue sectioning and analysis

[0132] The tumor and heart, liver, spleen, lung, kidney and other tissue sections can be used to determine the inhibitory effect of Pt-ATRA-TPP@TPGS on tumors and the side effects on mouse internal organs. Nude mice were killed, tumors and heart, liver, spleen, lung and kidney were removed, and each tissue was fixed for 12 hours with 10% paraformaldehyde solution. After dehydration, transparency, embedding and sectioning, HE staining (also known as hematoxylin-eosin staining) was performed on the tumor, heart, liver, spleen, lung and kidney tissues, and tunnel staining was performed on the tumor.

[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 a 5% CO2, 37°C cell culture incubator using HyClone1640 medium (containing 10% fetal bovine serum). After the cells grew to a certain number, the medium was removed and washed twice with PBS. After the cells were digested with trypsin for 1 min, serum-containing medium was added to terminate the digestion. After being blown evenly, the cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 2000 rpm for 5 min. The supernatant was discarded, PBS was added to wash 2 to 3 times, centrifuged, the supernatant was discarded, and PBS was added to make the cell concentration 2×10 7 / mL, mix well. Use a syringe to draw up the cell suspension, expel 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 assay

[0137] The mice injected with B16F10 cell suspension were injected with 30μM TPP, 10μM CiPt, 100μM ATRA, 150μg / mL TPGS, 10μM Pt-C12, and 10μM Pt-ATRA-TPP@TPGS, respectively, and three parallel experiments were set up. Another group was injected with saline only. After 21 days of observation, the mice were dissected, their lungs were removed, sliced ​​and photographed, and the melanoma in the mice's lungs was observed. At the same time, the lungs were 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 are combined under the action of van der Waals forces to form the inner core of the micelle, and the hydrophilic groups are outward to form the outer layer of the micelle, which is stably dispersed in the aqueous solution. However, cisplatin is soluble in water and is not easily carried by nano micelles. Therefore, the embodiment of the present invention modifies cisplatin by bonding lauric anhydride at both ends to make it insoluble in water. The nuclear magnetic resonance characterization of the synthesized Pt-C12 of the present invention is as follows Figure 1 As shown. It can be seen from 1HNMR that each peak of Pt-C12 prepared by the present invention has a clear attribution. Before bonding with lauric acid liver, the amino group of the platinum ligand is at 5.5ppm, and its peak becomes 6.5ppm after bonding. 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 embodiment of the present invention successfully synthesized Pt-C12.

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

[0142] The nanoparticles prepared in the present invention are characterized by transmission electron microscopy and field emission electron microscopy. Figure 2 and A and B, where Figure 2 A is the SEM image of Pt-ATRA-TPP@TPGS, and B is the TEM image of Pt-ATRA-TPP@TPGS. The results of transmission electron microscopy show that the synthesized Pt-ATRA-TPP@TPGS particles are relatively uniform in size, with a particle size of about 60nm, which is suitable for the use of 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 FIG. Before and after the addition of ATRA synthesized nanoparticles, the ultraviolet absorption of TPP is roughly the same, and there is no red shift or blue shift. At the same time, because the ultraviolet has absorption at 643nm, this also provides the possibility of using red light for cell experiments and in vivo experiments in the present invention. The drug release curve over time is shown in FIG. Figure 3 As shown in Figure B, the drug release increases with time.

[0144] The particle size distribution of Pt-ATRA-TPP@TPGS is shown in Figure 3 As shown in 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) Generation of ROS

[0146] Based on dimethyl-4-nitrosoaniline (N,N-Dimethyl-p-nitrosoaniline; RNO) as a selective receptor for singlet oxygen, in the presence of singlet oxygen, a decolorization effect is produced, and a decrease in the absorbance peak is shown 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 figure 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, but under light conditions, TPP and Pt-ATRA-TPP@TPGS had a bleaching effect on RNO, and the bleaching effect became more obvious as the light exposure time increased. This shows that ATRA replacing oleic acid did not affect the photodynamic efficiency of the particles.

[0147] In addition to RNO, methylene blue bleaching is also a common method for detecting hydroxyl radicals. Figure 5 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 figure is the illumination of Pt-ATRA-TPP@TPGS. Figure 5 It can be seen that under dark conditions, methylene blue was not bleached, but under 633nm light irradiation, methylene blue was bleached more and more with the increase of illumination time. This shows that under light conditions, TPP and Pt-ATRA-TPP@TPGS can both produce hydroxyl radicals, and the addition of ATRA has no negative effect 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 uses a DCFH-DA reactive oxygen species detection kit for testing, such as Figure 6 As shown, Figure 6 The results of singlet oxygen detection in cells are shown in Figure 2. In the dark, TPP and Pt-ATRA-TPP@TPGS have no fluorescence, indicating that neither of them produces singlet oxygen in the dark. However, in the light, they both emit green fluorescence, indicating that 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 produced by Pt-ATRA-TPP@TPGS after entering 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 detects cytotoxicity by MTT method. Figure 7 The cytotoxicity of the drugs to A549 and A549R cells was evaluated by MTT method. Figure 7 As shown in the figure, under 633nm light conditions, the toxicity of TPP and Pt-ATRA-TPP@TPGS to cells is significantly greater than that under dark conditions, which proves that TPP and Pt-TPP@TPGS have the effect of photodynamic therapy. At the same time, A549( Figure 7 A) and A549R ( Figure 7 Compared with the cells in B), Pt-ATRA-TPP@TPGS has better toxicity 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, live cells containing esterase after CalceinAM enters cells can produce Calcein, while dead cells cannot or rarely can produce Calcein, so only live 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, and dual fluorescent staining is carried out on live cells and dead cells at the same time, which can be used for the detection of cell activity and cytotoxicity.

[0154] like Figure 8 As shown, Figure 8 The live and dead cells were stained under the treatment of various drugs under dark conditions. 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 Fig. 9 As shown, Fig. 9 Live and dead cell staining of TPP and Pt-ATRA-TPP@TPGS under light and dark conditions. At the same concentration of TPP or Pt-ATR-TPP@TPGS, their cytotoxicity under light is greater than that under dark conditions, which proves the photodynamic therapy effect of TPP and Pt-ATR-TPP@TPGS.

[0156] (5) Anti-metastasis effect

[0157] In order to verify the anti-metastasis effect of Pt-ATR-TPP@TPGS, the present invention conducted a scratch experiment. The scratch experiment is a simple, fast and easy-to-observe experimental method for studying cell migration in vitro. Its principle is: when cells proliferate to the point of fusion into a monolayer state, an artificial blank area is created on the fused monolayer cells, called a "scratch". The cells on both sides of the scratch will gradually migrate into the blank area to heal the "scratch". It simulates the process of lateral cell migration in vivo to a certain extent.

[0158] like Fig.10 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 that nanomicelle-encapsulated ATRA has higher anti-metastasis ability than free ATRA.

[0159] As an experimental technique, Transwell mainly involves the Transwell chamber, which is placed in a culture plate. The inner part of the chamber is called the upper chamber, and the inner part of the culture plate is called the lower chamber. The upper and lower chambers are filled with culture fluids, respectively, and separated by a polycarbonate membrane. Cells are planted in the upper chamber. Because the polycarbonate membrane is permeable, the components in the culture fluid in the lower chamber can act on the cells in the upper chamber, thereby studying the effects of the culture fluid in the lower chamber on cell growth, proliferation, movement and migration. Using polycarbonate membranes with different pore sizes and different treatment pathways, various studies can be conducted on cell co-culture, cell chemotaxis, cell migration and invasion.

[0160] like Fig.11 It can be seen that there are basically no cells passing through the polycarbonate membrane of Pt-ATR-TPP@TPGS, which proves that Pt-ATR-TPP@TPGS can successfully inhibit cell migration. The migration inhibition ability of other components in the nanomicelles is shown in the figure.

[0161] The melanoma lung metastasis model of C57BL / 6J mice was established to lay the experimental foundation for the subsequent study of B16F10 anti-metastasis experiments. Fig.12 and 13 As shown, Fig.12 A mouse melanoma lung metastasis model. Fig.13 H&E staining of the lungs in a mouse melanoma lung metastasis model; during implantation of the B16F10 metastatic cell line, nanomicelle-encapsulated ATRA had a 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 selects the A549 subcutaneous tumor model to verify the tumor inhibition effect of Pt-ATRA-TPP@TPGS. The components set are: saline group, light group and dark group, so as to verify the photodynamic therapy effect of TPP material. Fig.14 shown. Fig.14 A in the figure represents the change of tumor volume over time; Fig.14 B in the figure represents the change of relative body weight of mice over time; Fig.14 C in the figure shows the tumor status of mice after treatment; Fig.14 D in is the tumor mass of mice; Fig.14 It can be seen that the tumor volume of the saline group was maintained at 400mm in the first five days after administration. 3Within 5 days, the tumor grew rapidly and was out of control. The tumor volume of mice treated with Pt-ATRA-TPP@TPGS was significantly inhibited compared with the saline group, regardless of light or darkness. Compared with the dark group, the light group had a better tumor inhibition effect, and the tumor in this group showed a trend of shrinking.

[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 Fig.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. Fig.16 The results of TUNEL staining of tumors. Fig.16 As shown, blue stains the cell nucleus and yellow indicates apoptosis. Fig.16 It can be seen that the saline group had no effect on the tumor, and the tumor tissues of the treatment groups suffered a certain degree of damage, especially the light-irradiated group. This shows that light enhanced the anti-tumor effect of the nano-micelles.

[0165] The present invention uses TPGS nano-micelles as carriers to co-transport Pt-C12, TPP, and ATRA to prepare an anti-metastatic nano drug Pt-ATRA-TPP@TPGS. In addition to having good chemotherapy and photodynamic therapy effects, the drug can also be used for anti-metastatic tumor treatment. The drug is dispersed more evenly in water, has good stability, has strong cytotoxicity, and exhibits excellent anti-metastatic effects. Pt-ATRA-TPP@TPGS has better anti-metastatic ability than free ATRA.

[0166] Although the above embodiment describes the present invention in detail, 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 protection scope of the present invention.

Claims

1. An anti-metastasis nano drug, characterized in that: The invention comprises vitamin E polyethylene glycol succinate nano-micelles, and lauric acid-modified tetravalent platinum prodrug, all-trans retinoic acid and a hydrophobic photosensitizer encapsulated in the vitamin E polyethylene glycol succinate nano-micelles; the lauric acid-modified tetravalent platinum prodrug is obtained by reacting Pt(IV)(OH)2 and lauric anhydride.

2. The anti-metastasis nano drug according to claim 1, characterized in that: The hydrophobic photosensitizer includes meta-tetraphenylporphine and / or hematophorin methyl ether.

3. The anti-metastasis nano drug according to claim 1, characterized in that: The particle size of the anti-metastasis nano drug is 60 nm.

4. The anti-metastasis nano drug 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(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; 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.

5. The method for preparing the anti-metastasis nano drug according to any one of claims 1 to 4, 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 material liquid is added dropwise to an inorganic strong alkali aqueous solution for mixing, and then the third organic solvent is removed to obtain the anti-transfer nano drug dispersion.

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

01.

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

5.

8. The preparation method according to claim 5 or 6, 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.

9. Use of the anti-metastatic nano drug according to any one of claims 1 to 4 or the anti-metastatic nano drug prepared by the preparation method according to any one of claims 5 to 8 in the preparation of anticancer drugs.

10. A chemotherapy-photodynamic combined anti-metastasis diagnostic and therapeutic agent, characterized in that: The invention comprises the anti-metastasis nano drug according to any one of claims 1 to 4 or the anti-metastasis nano drug prepared by the preparation method according to any one of claims 5 to 8.

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