Nano-drug, preparation method and application thereof, and chemotherapy-photodynamic combined diagnosis and treatment reagent

Abstract

The invention belongs to the technical field of anti-cancer drugs, and particularly relates to a nano-drug, a preparation method and application thereof, and a chemotherapy-photodynamic combined diagnosis and treatment reagent. The nano-drug provided by the invention comprises a vitamin E polyethylene glycol succinate nano-micelle, and a lauric acid modified tetravalent platinum prodrug, a hydrophobic photosensitizer and an emulsifier which are coated in the vitamin E polyethylene glycol succinate nano-micelle. According to the invention, co-transportation of the cis-platinum prodrug and the photosensitizer can be realized; after the 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; in the treatment process, the dosage of chemotherapeutic drugs is remarkably reduced, and side effects are greatly reduced.

Description

Technical Field

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

[0002] At present, clinical anti-cancer drugs are mainly platinum drugs. Platinum drugs induce apoptosis of cancer cells by inducing DNA damage. Its chemotherapy regimen is very effective and is widely used to treat a variety of malignant tumors. Because platinum drugs cannot specifically identify cancer cells, they can combine with normal molecules in the human body when treating cancer. They have strong toxic side effects and have great harm to the patient's organ function. Long-term use of platinum drugs will also produce drug resistance. One way to reduce side effects is to deliver drugs to the cancer site without leaking to other parts. This requires the drug delivery system to provide safe encapsulation of the drug before reaching the cancer site without leakage, but to be able to release the drug after entering the cancer tissue.

[0003] The selective delivery of platinum drugs to tumors by using nanocarriers is a common strategy that can reduce drug leakage into healthy tissues and increase drug levels in tumor tissues. Nanomicelle systems formed by self-assembly of surfactants or amphiphilic polymers in water can be used as carriers of hydrophobic drugs, macromolecular drugs, and gene drugs. When the shell of the micelle is composed of polyethylene glycol (PEG), it can effectively avoid the phagocytic effect of the reticuloendothelial system and prolong the circulation time of the drug in the body. At the same time, due to the rapid growth of tumor tissue, it has high permeability and retention for nanoparticles, namely the EPR effect, which allows the nanomicelles to be passively transported to the tumor site, so that the drugs at the tumor site are enriched, effectively reducing the toxic side effects of the drugs.

[0004] In addition to the passive targeting and reduced toxic side effects of nanomedicines, photosensitizers that respond to stimuli in the tumor microenvironment have also been widely studied. Photodynamic therapy (PDT) uses the interaction of photosensitizing drugs, light of appropriate wavelengths, and oxygen molecules to produce reactive oxygen species (ROS) to kill cancer cells. PDT can be used alone or in combination with surgery, chemotherapy, or ionizing radiation therapy. Photosensitizers are generally harmless in the absence of light, so tumor treatment can be precisely targeted by selective illumination, thereby limiting damage to surrounding healthy tissue.

[0005] However, currently available targeted drugs on the market cannot effectively achieve the simultaneous delivery of chemotherapeutic drugs and photosensitizers. Summary of the invention

[0006] The purpose of the present invention is to provide a nano drug and its preparation method and application, and a chemotherapy-photodynamic combined diagnostic and therapeutic reagent. The nano drug provided by the present invention can simultaneously deliver chemotherapy drugs and photosensitizers to tumor cells. The photosensitizer increases the production of singlet oxygen under the irradiation of red light, and works synergistically with the chemotherapy drugs to overcome drug resistance and achieve good therapeutic effects, and the side effects are greatly reduced.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The invention provides a nano drug, comprising vitamin E polyethylene glycol succinate nano micelles, and lauric acid-modified tetravalent platinum prodrug, a hydrophobic photosensitizer and an emulsifier 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.

[0009] Preferably, the emulsifier is one or more of oleic acid, Tween-80, stearic acid and vitamin E polyethylene glycol succinate.

[0010] Preferably, the hydrophobic photosensitizer includes m-tetraphenylporphine and / or hematophorin methyl ether.

[0011] Preferably, the average particle size of the nanomedicine is 40 to 50 nm.

[0012] Preferably, the method for preparing the lauric acid-modified tetravalent platinum prodrug comprises the following steps:

[0013] 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;

[0014] 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.

[0015] The present invention provides a method for preparing the nano drug described in the above technical solution, comprising the following steps:

[0016] mixing a lauric acid-modified tetravalent platinum prodrug, a hydrophobic photosensitizer, a third organic solvent and an emulsifier to obtain an emulsion;

[0017] dissolving vitamin E polyethylene glycol succinate in water to obtain a vitamin E polyethylene glycol succinate solution;

[0018] Mixing the emulsion and the vitamin E polyethylene glycol succinate solution to obtain a mixed liquid;

[0019] 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 nano drug dispersion.

[0020] Preferably, the molar ratio of the lauric acid-modified tetravalent platinum prodrug to the hydrophobic photosensitizer is 0.0025:0.01.

[0021] Preferably, the mass ratio of the lauric acid-modified tetravalent platinum prodrug to vitamin E polyethylene glycol succinate is 3.5:7.5.

[0022] The present invention provides the use of the nanomedicine described in the above technical solution or the nanomedicine prepared by the preparation method described in the above technical solution in the preparation of anticancer drugs.

[0023] The present invention provides a chemotherapy-photodynamic combined diagnostic and therapeutic reagent, including the nanomedicine described in the above technical solution or the nanomedicine prepared by the preparation method described in the above technical solution.

[0024] The invention provides a nano drug, comprising vitamin E polyethylene glycol succinate nano micelles, and lauric acid-modified tetravalent platinum prodrug, a hydrophobic photosensitizer and an emulsifier 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 present invention uses vitamin E polyethylene glycol succinate (abbreviated as vitamin E-TPGS or TPGS) under the action of an emulsifier to form a kinetically stable core-shell structure nano micelle as a drug carrier, and has a good encapsulation effect on a lauric acid-modified tetravalent platinum prodrug and a hydrophobic photosensitizer, and has good encapsulation and high drug loading, thereby realizing the co-transportation of cisplatin prodrug and photosensitizer; after the nano drug 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 cooperate with the chemotherapeutic drug (tetravalent platinum) to combine chemotherapy and photodynamic therapy, thereby overcoming drug resistance and achieving good therapeutic effects; during the treatment process, the dosage of the chemotherapeutic drug is significantly reduced, and the side effects will be greatly reduced.

[0025] 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

[0026] Figure 1 The hydrogen nuclear magnetic resonance spectrum of Pt-C12 prepared in Example 1;

[0027] Figure 2 The average particle size of nanomicelles, TPP encapsulation efficiency and Pt encapsulation efficiency when different emulsifiers are used in Examples 1 to 3;

[0028] Figure 3 TEM image and SEM image of Pt-TPP@TPGS prepared in Example 1;

[0029] Figure 4 The zeta potential of Pt-TPP@TPGS prepared in Example 1 at different pH values;

[0030] Figure 5 The UV absorption spectra of TPP and Pt-TPP@TPGS (TPP concentration is 10 μM) and the release rate of TPP in Pt-TPP@TPGS over time;

[0031] Figure 6 The fluorescence test results of EB and DNA embedding;

[0032] Figure 7 is the singlet oxygen detection result;

[0033] Figure 8 It is the result of Fenton reaction test;

[0034] Fig. 9 The DCFH-DA reactive oxygen species detection kit detects the production of singlet oxygen in cells;

[0035] Fig.10 Confocal microscopy images of A549 cells after incubation with Pt-TPP@TPGS (10 μM) for 1 h and co-treatment with lysosomal green probe (5 μM, 2 h) and mitochondrial green probe (150 nM, 30 min);

[0036] Fig.11 To study the cytotoxicity of the complex;

[0037] Fig.12 Calcein-AM / PI kit for live and dead cell staining (1 μM);

[0038] Fig.13 for live and dead cell staining (Pt concentration 1 μM, TPP concentration 3 μM);

[0039] Fig.14 The tumor inhibition effect and photodynamic therapy effect of Pt-TPP@TPGS;

[0040] Fig.15 H&E staining results for heart, liver, spleen, lung, kidney, and tumor;

[0041] Fig.16 This is the TUNEL staining result of the tumor;

[0042] Fig.17 The synthetic route of lauric acid-modified tetravalent platinum prodrug Pt-C12. DETAILED DESCRIPTION

[0043] The invention provides a nano drug, comprising vitamin E polyethylene glycol succinate nano micelles, and lauric acid-modified tetravalent platinum prodrug, a hydrophobic photosensitizer and an emulsifier 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.

[0044] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0045] The nano medicine provided by the invention comprises vitamin E polyethylene glycol succinate nano micelles.

[0046] The chemical structural formula of the vitamin E polyethylene glycol succinate provided by the present invention is shown in Formula 1:

[0047]

[0048] 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) under the action of an emulsifier, and exhibits the properties of a solubilizer, emulsifier, dispersant and gelling agent, and can regulate its aggregation behavior together with other amphiphiles and organic additives.

[0049] 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 repulsion between water molecules and TPGS is stronger than the attraction. The hydrophobic groups associate under the action of van der Waals forces 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.

[0050] The 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.

[0051] In the present invention, the method for preparing the lauric acid-modified tetravalent platinum prodrug preferably comprises the following steps:

[0052] 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;

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] In the present invention, the chemical structure of the lauric acid-modified tetravalent platinum prodrug is shown in Formula 2:

[0060]

[0061] The nano medicine provided by the invention comprises a hydrophobic photosensitizer encapsulated in the vitamin E polyethylene glycol succinate nano micelle.

[0062] In the present invention, the hydrophobic photosensitizer includes m-tetraphenylporphine (TPP) and / or hematophorin methyl ether, more preferably m-tetraphenylporphine (TPP).

[0063] In the present invention, the chemical structural formula of the TPP is shown in Formula 3:

[0064]

[0065] In the present invention, the average particle size of the nano drug is preferably 40 to 50 nm.

[0066] In the nanomedicine provided by the present invention, the mass ratio of the lauric acid-modified tetravalent platinum prodrug to the hydrophobic photosensitizer is preferably 7:9.

[0067] The nano medicine provided by the invention comprises an emulsifier coated in the vitamin E polyethylene glycol succinate nano micelle.

[0068] In the present invention, the emulsifier is preferably one or more of oleic acid, Tween-80, stearic acid and vitamin E polyethylene glycol succinate, more preferably oleic acid.

[0069] In the nanomedicine provided by the present invention, the mass ratio of the vitamin E polyethylene glycol succinate nanomicelles to the emulsifier is preferably 5:2.

[0070] The present invention provides a method for preparing the nano drug described in the above technical solution, comprising the following steps:

[0071] mixing a lauric acid-modified tetravalent platinum prodrug, a hydrophobic photosensitizer, a third organic solvent and an emulsifier to obtain an emulsion;

[0072] dissolving vitamin E polyethylene glycol succinate in water to obtain a vitamin E polyethylene glycol succinate solution;

[0073] Mixing the emulsion and the vitamin E polyethylene glycol succinate solution to obtain a mixed liquid;

[0074] The mixed material liquid is added dropwise into an inorganic strong alkali aqueous solution to obtain a dispersion of the nano drug.

[0075] The present invention mixes a lauric acid-modified tetravalent platinum prodrug, a hydrophobic photosensitizer, a third organic solvent and an emulsifier to obtain an emulsion. In the present invention, the emulsifier is preferably one or more of sodium oleate, Tween-80, stearic acid and vitamin E polyethylene glycol succinate, more preferably sodium oleate. The third organic solvent is specifically preferably chloroform. In the present invention, the molar ratio of the lauric acid-modified tetravalent platinum prodrug and the hydrophobic photosensitizer is 0.0025:0.01. The molar ratio of the lauric acid-modified tetravalent platinum prodrug and the emulsifier is preferably 0.005:0.1.

[0076] 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 emulsifier in the remaining third organic solvent to obtain an emulsifier solution; mixing the lauric acid-modified tetravalent platinum prodrug solution, the photosensitizer solution and the emulsifier 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 emulsifier solution is preferably 100mmol / L; when the lauric acid-modified tetravalent platinum prodrug solution, the photosensitizer solution and the emulsifier solution are mixed, the volume ratio of the lauric acid-modified tetravalent platinum prodrug solution, the photosensitizer solution and the emulsifier solution is preferably 2:2:1.

[0077] 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.

[0078] After obtaining the emulsion and the vitamin E polyethylene glycol succinate solution, the present invention mixes the emulsion and the vitamin E polyethylene glycol succinate solution to obtain a mixed liquid.

[0079] 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 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 0.5:1.1. 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 1h, until the third organic solvent is volatilized.

[0080] The nano drug dispersion provided by the present invention is preferably an aqueous dispersion of the nano drug. In the present invention, the molar concentration of Pt in the aqueous dispersion of the nano drug is preferably 98.8 μM, and the encapsulation efficiency is as high as 98.8%. The TPP content can be measured by UV-visible spectrophotometer, and the TPP concentration is preferably 305 μM, and the encapsulation efficiency is 76.25%. It can be calculated that the TPGS concentration is preferably 1.5 mg / mL, and the OA (sodium oleate) concentration is preferably 2 mM. In the present invention, the encapsulation efficiency = the drug inside the nano drug particle / the drug added during the synthesis process × 100%.

[0081] The present invention provides the use of the nanomedicine described in the above technical solution or the nanomedicine prepared by the preparation method described in the above technical solution in the preparation of anticancer drugs.

[0082] The present invention provides a chemotherapy-photodynamic combined diagnostic and therapeutic reagent, including the nanomedicine described in the above technical solution or the nanomedicine prepared by the preparation method described in the above technical solution.

[0083] 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.

[0084] The experimental reagents and instruments used in the following examples are shown in Table 1 and Table 2.

[0085] Table 1 Experimental reagents and manufacturers

[0086]

[0087]

[0088] Table 2 Main experimental instruments

[0089]

[0090]

[0091] Example 1

[0092] according to Fig.17 Flow chart: 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%.

[0093] Weigh Pt(Ⅳ)(C12)2 (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(Ⅳ)(C12)2; 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 oleic acid (OA, 28.2 mg, 0.1 mmol) in 1 mL of chloroform to obtain a 100 mmol / L oleic acid chloroform solution. Take 200 μL of the above-prepared Pt(Ⅳ)(C12)2 solution, 200 μL TPP solution, and 100 μL oleic acid solution, mix them together, and adjust the volume to 1 mL. In addition, prepare a 1 mol / L sodium hydroxide solution for use.

[0094] 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 then added dropwise to 11 μL, 1 mol / L NaOH solution, and ultrasonically vibrated for 1 hour in a 40°C water bath 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-TPP@TPGS. After determination by inductively coupled plasma mass spectrometry (ICP-MS), the Pt concentration was calculated to be 98.8 μM, and the encapsulation efficiency was as high as 98.8%. The TPP content was determined by UV-visible spectrophotometer, and the TPP concentration was 305 μM, and the encapsulation efficiency was 76.25%. It was calculated that the TPGS concentration was 1.5 mg / mL and the OA concentration was 2 mM.

[0095] Example 2

[0096] The preparation method is basically the same as that of Example 1, except that Tween-80 is used to replace the oleic acid in Example 1 as the emulsifier.

[0097] Example 3

[0098] The preparation method is basically the same as that of Example 1, except that stearic acid is used to replace the oleic acid in Example 1 as the emulsifier.

[0099] Comparative Example 1

[0100] The preparation method is basically the same as that of Example 1, except that the amount of oleic acid used is 0 mg.

[0101] The performance test is carried out below using the product Pt-TPP@TPGS prepared in Example 1 as an example.

[0102] Test Example 1

[0103] (1) Particle size distribution and stability study

[0104] The particle size distribution of Pt-TPP@TPGS was measured using a dynamic light scattering instrument. During the measurement, 20 μL of the prepared nanoparticles Pt-TPP@TPGS 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.

[0105] (2) Particle morphology observation

[0106] The prepared nanoparticles were diluted 10 times and then dripped onto a 230-mesh copper grid containing a carbon film using a capillary tube, and the particle morphology was observed using a transmission electron microscope. A small amount of the liquid was also pipetted onto a silicon wafer using a pipette, and the particle morphology was observed using a SEM after drying.

[0107] (3) Drug release experiment

[0108] 1 mL of prepared Pt-TPP@TPGS nanoparticles was taken into a dialysis bag (1000 molecular weight), the dialysis bag was sealed and placed in 10 mL of PBS buffer, and dialyzed for different time periods (0, 1, 2, 4, 8, 12, 24 hours). Then, 200 μL of the dialyzed solution was taken to measure the UV absorption, and 200 μL of buffer was added to the dialysis beaker to ensure that the volume of the dialyzed liquid remained unchanged. The drug release rate was determined by measuring the concentration of released TPP.

[0109] (4) Hydroxyl radical detection

[0110] Measure the generation of hydroxyl radicals by detecting the amount of methylene blue bleaching

[102] The specific detection steps are as follows: TPP and Pt-TPP@TPGS solutions (TPP concentration: 10 μM) were mixed with methylene blue solution (12.5 mg / L) respectively, and then the mixture was illuminated with 633 nm light (30 mW / cm 2 ) were irradiated for different time periods (0, 5, 10, 15, 20, 25, 30 min), and the absorbance of methylene blue was measured using a UV-visible spectrophotometer.

[0111] (5) Reduction of Pt-C12

[0112] Ethidium bromide (EB) is a fluorescent dye commonly used to observe DNA under ultraviolet light. It has the ability to specifically bind to nucleic acids (DNA, RNA, double-stranded polynucleotides). The minimum detection amount of deoxyribonucleic acid can reach 10ng / mL, which is very sensitive.

[0113] The way cisplatin kills cancer cells is that cisplatin interacts with purine bases to produce irreparable platinum-DNA adducts, which ultimately lead to sufficient DNA damage to cause cell apoptosis. The already damaged DNA does not react with ethidium bromide.

[0114] Prepare 2mL 4mg / mL fish sperm DNA, 2mL 2 mM Pt-C12 (dissolved in DMF), 2mL 2mM cisplatin, and 2mL 100mM ascorbic acid for later use. Dilute 4mg / mL fish sperm DNA to 0.4mg / mL, add 200μL of the prepared fish sperm DNA solution, 100μLPt-C12, 100μL ascorbic acid, and then add 600μL of water and 1000μL of PB buffer (without sodium ions) to a 5mL centrifuge tube to make the total volume 2000μL. After a certain time interval (0, 0.5, 1, 2, 4, 8, 12, 24h), take out 200μL from the mixed solution system prepared above and place it in a 1.5mL centrifuge tube and freeze it in a -80℃ refrigerator.

[0115] In addition, 200 μL of the prepared fish sperm DNA solution, 100 μL of CiPt, 100 μL of ascorbic acid, 600 μL of water and 1000 μL of PB buffer (without sodium ions) were added to a 5 mL centrifuge tube to make the total volume 2000 μL. After a certain time interval (0, 0.5, 1, 2, 4, 8, 12, 24 h), 200 μL was taken out from the mixed solution system prepared above and placed in a 1.5 mL centrifuge tube and stored in a -80°C refrigerator. Negative and positive controls were also performed for this experiment.

[0116] Prepare 1mg / mL ethidium bromide solution and dilute it 20 times with PB (sodium-free) buffer to obtain 0.05mg / mL ethidium bromide solution. Add 200μL ethidium bromide solution to the frozen reaction solution from time 0 according to the time gradient, mix well and measure its fluorescence immediately. The excitation wavelength and emission wavelength of ethidium bromide are: Ex: 530nm; Em: 592nm.

[0117] The amount of remaining DNA in the reaction solution was determined based on the intensity of fluorescence, and the reaction rates of CiPt and Pt-C12 with DNA were reversed.

[0118] (6) Cell culture

[0119] Take out the frozen cells from the -80℃ refrigerator, thaw them quickly and transfer them to a 15mL centrifuge tube. Centrifuge at 2000rpm for 5min, remove the supernatant, add 2mL of serum-containing culture medium to the cell pellet, disperse evenly and transfer the cell suspension to a 25cm 3 In the culture medium, replace the culture medium after standing overnight, and pass the cells after they are fully grown. When passing the cells, wash the cells to be treated twice with 2mL PBS buffer, add 1mL trypsin to digest the cells, put them in the incubator for digestion for about 20 seconds, wait for the cell morphology to change under the microscope, and the cells begin to become spherical from the adherent state, quickly remove the trypsin, add 2mL of culture medium containing serum, blow the cells on the wall of the culture bottle evenly and transfer them to a sterile centrifuge tube, centrifuge at 2000rpm for 5min, remove the supernatant, add an appropriate amount of culture medium to resuspend, and transfer the cell suspension that meets the experimental needs to a 25cm 2 Place the culture bottle in the incubator for further culture or for various experiments.

[0120] (7) Cytotoxicity test

[0121] The MTT method, also known as the MTT colorimetric method, is a method for detecting cell survival and growth. The succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan and deposit it in the cells, while dead cells do not have this function. After dissolving the crystals with DMSO, the cytotoxicity of the drug is analyzed by measuring the absorbance at 490nm with an enzyme marker.

[0122] 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 incubated for 1 h. After completion, the plates were treated in darkness and 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 for dissolution, shake it on a shaker and detect the absorbance of each well at 490nm. IC 50 The values ​​were calculated using GraphPad Prism software (Equation 1).

[0123]

[0124] Test Example 2

[0125] (1) ROS detection

[0126] Singlet oxygen quantum yield determination

[0127] The samples TPP and Pt-TPP@TPGS to be tested were 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 633nm surface light source (30mW / cm 2 ) and record the absorbance of RNO at 440nm after 5min, 10min, 15min, 20min, 25min, and 30min of illumination to obtain the absorbance change curve under different illumination time. At the same time, a negative control was performed under dark conditions.

[0128] Intracellular ROS detection

[0129] 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 4The 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-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.

[0130] (2) Laser confocal microscopy

[0131] Appropriate A549 cells were inoculated into laser confocal culture dishes, and 5μM nanoparticles Pt-TPP@TPGS were added after adherent growth. After incubation for 1 hour, 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 to 3 times with serum-free culture medium and PBS, and observed with a Zeiss LSM 880 confocal microscope (63× oil lens).

[0132] (3) Live / dead cell staining experiment

[0133] 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, 20 μM OA, 15 μg / mL TPGS, 1 μM Pt-C12, and 1 μM Pt-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-TPP@TPGS were illuminated with a 633 nm surface light source (30 mW / cm 2 , 30min), placed in an incubator for 24h, and then stained and observed.

[0134] Test Example 3: Tumor Inhibition Experiment

[0135] Establishment of animal model

[0136] 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, for a total of 15 mice. Observe the growth of nude mice, weigh them regularly, and measure the size of tumors in nude mice.

[0137] In vivo tumor inhibition experiment

[0138] 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-TPP@TPGS. 8h after the injection of the drug, the light group mice were irradiated with a 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.

[0139] Tissue sectioning and analysis

[0140] The tumor inhibition effect of Pt-TPP@TPGS and the side effects on the mouse internal organs can be determined by sections of tumors and heart, liver, spleen, lung, kidney and other tissues. 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.

[0141] Test Results

[0142] (1) Structural characterization of the complex

[0143] 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. 1 HNMR shows that each peak of Pt-C12 prepared by the present invention has a clear attribution. Before bonding with lauric acid, the amino group of the platinum ligand is at 5.5ppm, and after bonding, its peak becomes 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.

[0144] In the process of preparing nanoparticles, Examples 1 to 3 respectively use sodium oleate, Tween-80 and stearic acid as emulsifiers. The effects of the products prepared in Examples 1 to 3 and Comparative Example 1 are as follows: Figure 2 shown. Figure 2 A in is the nanomicelle particle size when using different emulsifiers; Figure 2 B in the figure is the TPP encapsulation efficiency when using different emulsifiers; Figure 2 C is the Pt encapsulation rate when using different emulsifiers. After comparing the particle size and drug encapsulation rate, the present invention decided to use sodium oleate as the emulsifier, the synthesized nano micelles have a smaller average particle size, a higher drug encapsulation rate, and are more conducive to biological experiments.

[0145] The nanoparticles prepared in the present invention are characterized by transmission electron microscopy and field emission electron microscopy. Figure 3 As shown. The transmission electron microscopy results show that the Pt-TPP@TPGS particles synthesized in Example 1 are relatively uniform in size, with an average particle size of about 50 nm, which is consistent with the average particle size measured by the Malvern potentiostat, and the size is suitable for the use of nanomedicine. The present invention uses a dynamic light scattering instrument to study the zeta potential of the Pt-TPP@TPGS prepared in Example at different pH values. When pH = 6.8, the zeta potential is -5.13; when pH = 7.2, the zeta potential is -6.74; in DMEM, the zeta potential is -5.89, as shown in FIG. Figure 4 The Pt-TPP@TPGS product prepared in Example 1 has a lower potential, which is conducive to the uptake of nano-micelles by cells.

[0146] (2) Drug mechanism research

[0147] In order to understand the ultraviolet absorption of TPP before and after being made into nanoparticles, the present invention conducted ultraviolet tests on TPP and Pt-TPP@TPGS prepared in Example 1. Figure 5 As shown in A and B in FIG. Before and after being made into nanoparticles, TPP has ultraviolet absorption at 410nm, 509nm, 545nm, 585nm, and 643nm, and there is no obvious red shift or blue shift in the ultraviolet absorption. The absorption at 643nm means that the present invention can use red light with stronger penetrating power for cell experiments and in vivo experiments.

[0148] In order to verify the drug release, the present invention determines the concentration of the precipitated drug by measuring the ultraviolet TPP of the dialysate. Figure 5 As shown in Figure C, the concentration of TPP in the external fluid increases with the increase of dialysis time, which proves that the nanomicelles can release drugs over time.

[0149] The present invention measures the fluorescence of EB and DNA embedding to obtain the embedding amount of EB and DNA, and simultaneously infers the reaction rate of cisplatin and DNA. Figure 6 As shown in A, after the reaction of cisplatin and DNA for a certain period of time, the fluorescence of the remaining DNA and EB in the centrifuge tube weakened with time, indicating that the interaction between cisplatin and the purine bases on DNA produced an irreparable platinum-DNA adduct. The Pt-C12 prepared in Example 1 also reacted with DNA in a 100mM ascorbic acid environment, which also proved that the Pt-C12 prepared in Example 1 can be reduced by ascorbic acid to become cisplatin, and the cisplatin it produced has reacted with DNA. Figure 6 The reaction rate of cisplatin with Pt-C12 (ascorbic acid) and DNA is shown in B. Figure 6 As shown in C, Figure 6 The emission intensity of the vertical coordinates in A and B ranges from 0 to 2.5×10 5 .

[0150] In order to verify the micelle stability of the nano drug Pt-TPP@TPGS prepared in Example 1, the particle size of the nano micelles was measured using a Malvern potentiostat. Figure 6 As shown in D in the figure, as time changes, the average particle size of Pt-TPP@TPGS is always around 40-50 nm, which indicates that the nanomicelles are relatively stable in both water and DMEM.

[0151] (3) Singlet oxygen detection

[0152] 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 7 As shown, Figure 7 A in the figure is the detection of RNO / His reagent and the generation of different drugs under 633nm light 1 O2 (A) TPP changes over time under dark conditions; Figure 7 B in the figure is the change of TPP with illumination time under illumination conditions; Figure 7 C in the figure is the change of Pt-TPP@TPGS over time under dark conditions; Figure 7 D in the figure is the change of Pt-TPP@TPGS under illumination conditions with illumination time.

[0153] Depend on Figure 7 It can be seen that: under dark conditions, whether it is TPP or Pt-TPP@TPGS, the ultraviolet absorption of RNO does not change, proving that they do not produce singlet oxygen, but under light conditions, TPP and Pt-TPP@TPGS prepared in Example 1 both have a bleaching effect on RNO, and the bleaching effect steadily decreases with the extension of the light exposure time.

[0154] Fenton reaction is one of the methods to detect hydroxyl free radicals. After hydroxyl free radicals react with methylene blue, the color of methylene blue changes. The change of its absorption peak can be measured by UV-spectrophotometer to indirectly measure the amount of hydroxyl free radicals produced. Figure 8 As shown, Figure 8 For methylene blue detection and hydroxyl radicals generated by different drugs under 633nm light, Figure 8 The A in it stands for TPP, dark, Figure 8 B in the equation is TPP, light, Figure 8 C in the figure is Pt-TPP@TPGS, dark, Figure 8 D in is Pt-TPP@TPGS, light. Figure 8 It can be seen that: under dark conditions, neither TPP nor Pt-TPP@TPGS bleached methylene blue, and the absorption peak intensity did not change, proving that they did not produce hydroxyl radicals. However, under light conditions, both TPP and Pt-TPP@TPGS bleached methylene blue, and as the light exposure time increased, the amount of hydroxyl radical bleaching increased, proving that they both produced hydroxyl radicals under light conditions.

[0155] In order to verify the singlet oxygen generated by TPP and Pt-TPP@TPGS after entering the cells, the present invention uses DCFH-DA fluorescent probe to detect the ROS generated by TPP and Pt-TPP@TPGS in the cells, such as Fig. 9 As shown, Fig. 9 The DCFH-DA reactive oxygen species detection kit was used to detect the production of singlet oxygen in cells. Under dark conditions, TPP and Pt-TPP@TPGS prepared in Example 1 did not produce ROS, but under light conditions, TPP and Pt-TPP@TPGS were able to effectively produce ROS. In addition, when the TPP concentration was 30 μM, the singlet oxygen fluorescence produced by Pt-TPP@TPGS after entering the cells was stronger than that produced by the TPP group, which also shows that the TPP loaded by the nanoparticles entered the cells more than the free TPP.

[0156] (4) Cell co-localization experiment

[0157] The cells showed significant uptake effect and strong imaging results for the drug Pt-TPP@TPGS. In order to accurately obtain the location information of the complex, the present invention selected the lysosomal dye Lyso-Tracker and Mito-Tracker Co-localization experiments with Pt-TPP@TPGS, such as Fig.10 , Fig.10 Confocal microscopy images of A549 cells treated with lysosomal green probe (5 μM, 2 h) and mitochondrial green probe (150 nM, 30 min) after incubation with Pt-TPP@TPGS (10 μM) for 1 h. Fig.10 It can be clearly seen that the Pt-TPP@TPGS prepared in Example 1 highly overlaps with the lysosomal dye, indicating that the complex selectively accumulates in the cell lysosome and has a good cell imaging effect.

[0158] (5) Cytotoxicity

[0159] The cytotoxicity of the drugs on A549 and A549R cells was evaluated by MTT assay. Fig.11 As shown, Fig.11 To study the cytotoxicity of the complex, Fig.11 A in the figure represents A549 cells incubated with different concentrations of drugs for 1 h and irradiated with 633 nm light (30 mW / cm 2 ); Fig.11 B shows A549R cells incubated with different concentrations of drugs for 1 h and irradiated with 633 nm light (30 mW / cm 2 ). Fig.11It can be seen that under 633nm light conditions, the toxicity of TPP and Pt-TPP@TPGS to cells is significantly greater than that under dark conditions, which proves that TPP and Pt-TPP@TPGS have excellent photodynamic therapy effects. Fig. 9 A) and A549R ( Fig. 9 B) In both cells, Pt-TPP@TPGS can inhibit the growth of cancer cells better than cisplatin.

[0160] The following are the IC values ​​calculated by GraphPadPrism software: 50 The values ​​are shown in Table 3.

[0161] Table 3 IC of each drug 50 value

[0162]

[0163] In order to more intuitively show the difference in the therapeutic effect of drugs on cancer cells, the present invention uses Calcein-AM / PI live / dead cell co-staining reagent to test the actual therapeutic effect of cells. Calcein-AM can stain live cells green, and PI can stain dead cells red. The results are as follows: Fig.12 shown. Fig.12 Live and dead cells were stained using Calcein-AM / PI kit (1 μM).

[0164] In order to more intuitively demonstrate the photodynamic therapy effect of TPP and Pt-TPP@TPGS, the present invention also stains the TPP and Pt-TPP@TPGS cells in the dark group and the light group after drug treatment. Fig.13 Live and dead cell staining (Pt concentration 1μM, TPP concentration 3μM). The present invention significantly found that, whether it is TPP or Pt-TPP@TPGS, the cell lethality in the light group is higher than that in the dark group, which is consistent with the cytotoxicity, once again proving the photodynamic therapy effect of the nanodrug Pt-TPP@TPGS.

[0165] (6) Tumor inhibition experiment

[0166] In order to prove that the prepared nanomedicine has good effects at the animal level, the present invention selects the A549 subcutaneous tumor model to verify the tumor inhibition effect and photodynamic therapy effect of Pt-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 As shown, Fig.14A in the figure shows the growth of mouse tumors, including the blank group, Pt-TPP@TPGS Irradiation group, and Pt-TPP@TPGSDark group; Fig.14 B in the figure shows the morphology of mouse tumor after treatment; Fig.14 C in the figure is the growth of subcutaneous tumor volume in mice; Fig.14 D in the figure is the relative weight change of mice during treatment. The tumor volume of the saline group was maintained at 400 mm in the first five days after administration. 3 Within 5 days, the tumor grew rapidly and was out of control. The tumor volume of mice treated with Pt-TPP@TPGS was significantly inhibited compared with the saline group, regardless of whether they were exposed to light or darkness. Compared with the dark group, the tumor inhibition effect of the light group was better, and the tumor size of this group hardly changed, maintaining a relatively small level (about 200mm 3 ),like Fig.14 As shown in A, B and C. Meanwhile, during the tumor inhibition process, the weight of the mice was monitored by the present invention, as shown in FIG. Fig.14 As shown in D. The body weight of mice increases with the age of mice and the growth of mouse tumors.

[0167] In addition, in order to further verify the toxicity and safety of the nanomedicine Pt-TPP@TPGS, the present invention conducted a series of studies on various tissue sections and tumor sections of mice. Fig.15 (H&E staining of heart, liver, spleen, lung, kidney, and tumor). From the H&E-stained sections, it can be seen that compared with the saline group, there is no obvious necrosis in the organs and tissues of the mice with Pt-TPP@TPGS, which proves that the drug has low toxicity and side effects. At the same time, H&E staining of the tumor site shows that the tumor with Pt-TPP@TPGS has obvious necrosis compared with the saline group, which proves the therapeutic effect of Pt-TPP@TPGS.

[0168] At the same time, in order to further study the tumor inhibition mechanism, the present invention performed TUNEL staining on tumor tissue sections. Fig.16 (TUNEL staining of tumor) shows that 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.

[0169] The present invention uses the function of amphiphilic polymer chains to self-assemble in water to form nano micelles, which encapsulate two drugs, namely, chemotherapy drugs, tetravalent platinum prodrug Pt-C12 and photodynamic therapy drug TPP, to form Pt-TPP@TPGS nanoparticles. The nanoparticles have high stability and are evenly distributed in aqueous solution. The reaction of Pt-C12 with DNA under the action of ascorbic acid proves that the tetravalent platinum prodrug can be reduced under high reduction conditions in cancer cells. The nano drug can generate active oxygen during the treatment process. Through cytotoxicity and other tests, the drug can effectively overcome the drug resistance of cancer cells. It is also proved at the animal level that the material has a good tumor inhibition effect.

[0170] 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. A nano drug, characterized in that: It includes vitamin E polyethylene glycol succinate nano-micelles, and lauric acid-modified tetravalent platinum prodrug, hydrophobic photosensitizer and emulsifier encapsulated in the vitamin E polyethylene glycol succinate nano-micelles; the lauric acid-modified tetravalent platinum prodrug is obtained by the reaction of Pt(IV)(OH)2 and lauric anhydride.

2. The nano drug according to claim 1, characterized in that The emulsifier is one or more of oleic acid, Tween-80, stearic acid and vitamin E polyethylene glycol succinate.

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

4. The nano drug according to claim 1, characterized in that The average particle size of the nano drug is 40 to 50 nm.

5. The 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.

6. The method for preparing the nano drug according to any one of claims 1 to 5, characterized in that: The following steps are involved: mixing a lauric acid-modified tetravalent platinum prodrug, a hydrophobic photosensitizer, a third organic solvent and an emulsifier to obtain an emulsion; dissolving vitamin E polyethylene glycol succinate in water to obtain a vitamin E polyethylene glycol succinate solution; Mixing the emulsion 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 nano drug dispersion.

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

01.

8. The preparation method according to claim 6 or 7, 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 nanomedicine according to any one of claims 1 to 5 or the nanomedicine prepared by the preparation method according to any one of claims 6 to 8 in the preparation of anticancer drugs.

10. A chemotherapy-photodynamic combined diagnostic and therapeutic reagent, characterized in that: The invention comprises the nano drug according to any one of claims 1 to 5 or the nano drug prepared by the preparation method according to any one of claims 6 to 8.

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