A kind of hydroxyalkyl starch-photosensitizer coupled amphiphilic macromolecular compound, nano drug delivery system and its preparation method and application

Abstract

The present application belongs to the technical field of chemistry, pharmacy, medicine and other multi-disciplinary, more specifically, a kind of hydroxyalkyl starch-photosensitizer coupled amphiphilic macromolecular compound, nano drug delivery system and its preparation method and application are disclosed.The present application uses hydrophobic porphyrin heterocyclic macrocyclic organic compound photosensitizer as the hydrophobic end, coupled with hydroxyalkyl starch, and assembled into nanoparticles with hydroxyalkyl starch-platinum (IV) conjugate.The experiment found that the amphiphilic macromolecular compound can improve the water solubility of photosensitizer, and also can maintain the photodynamic characteristics of photosensitizer, give its targeting tumor characteristics, improve the bioavailability of photosensitizer, and the added hydroxyalkyl starch-platinum (IV) conjugate can release cisplatin in vivo, inhibit the activity of thioredoxin reductase, destroy the tumor redox balance, promote the effect of photodynamic therapy, realize the targeted combination therapy of antitumor drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of chemistry, pharmacy, medicine and the like, and more particularly relates to a hydroxyalkyl starch-photosensitizer coupled amphiphilic macromolecular compound, a nano drug delivery system and a preparation method and application thereof. BACKGROUND

[0002] Photodynamic therapy (PDT) is a very important modern tumor treatment method, which has the advantages of minimal invasion, good targeting and small adverse reactions. Photodynamic therapy is composed of photosensitizer, oxygen and light, and the three work together to produce reactive oxygen species, thereby killing tumors and achieving the effect of resisting tumors. Photosensitizer (PS) is an organic or inorganic substance with good stability and no dark toxicity, which will produce singlet oxygen and other strong oxidizing molecules under the irradiation of light of a specific wavelength. However, most photosensitizers, such as pyropheophorbide-a, are water-insoluble and non-specific, which limits their application in tumor treatment. Moreover, it is reported that the efficacy of photodynamic therapy is inhibited by intracellular high reducing power, which affects the effect of photodynamic therapy.

[0003] Chemotherapy is one of the most effective means of treating tumors at present. Although many common chemotherapeutic drugs have certain therapeutic effects, there are still great defects in a large number of marketed chemotherapeutic drugs. The selectivity of divalent platinum drugs for tumors mainly comes from the high demand of tumors for nutrients, but this selectivity is very weak, and other rapidly growing tissues (such as bone marrow, hair follicles, etc.) will also produce toxicity due to increased uptake; at the same time, Pt(II) drugs are often cleared through the kidneys, thus also producing certain hepatotoxicity and nephrotoxicity. In addition, Pt(II) drugs are unstable, and they are prone to react with nucleophiles (especially human serum albumin) in the blood circulation, resulting in reduced bioavailability and increased toxic side effects. Tetravalent platinum prodrugs are low-spin octahedral complexes obtained by oxidizing addition of two axial ligands to divalent platinum, and the octahedral structure of tetravalent platinum prodrugs makes them less likely to react with other ligands, but they can be reduced by reducing agents to release two axial ligands, thereby releasing the original divalent platinum drugs. This makes tetravalent platinum prodrugs stable in the blood circulation, but rapidly converted into divalent platinum in cells to exert their effects. Tetravalent platinum complexes can reduce the toxic side effects of divalent platinum, but like small molecules, they also have some problems in drug delivery, such as poor water solubility, too fast blood clearance, insufficient accumulation at tumor sites and low bioavailability, etc.

[0004] With the development of science and technology in recent decades, nanocarrier has become one of the effective means to solve the above problems. At present, a variety of drug-loaded nanocarriers have entered the market, and a large number of nanocarriers are in the clinical research and preclinical research stage. Amphiphilic polymer drug-loaded nanosystem is one of the nanocarriers that are currently studied more. It can provide a hydrophobic core to solubilize hydrophobic drug molecules, and its hydrophilic shell can reduce protein adsorption and reduce the phagocytic clearance of the reticuloendothelial system, prolonging the half-life of the drug in the body.

[0005] However, in the actual construction process of the amphiphilic nanocarrier, although the technical problems of poor water solubility of photosensitizers and chemotherapeutic drugs and too fast blood clearance speed can be solved to some extent, the nanocarrier prepared often reduces the photodynamic therapy effect of the photosensitizer itself or the amount of drug uptake at the tumor site is insufficient, resulting in poor tumor killing effect of the nanocarrier. SUMMARY

[0006] In view of the defects of the prior art, the purpose of the present application is to provide a hydroxyalkyl starch-photosensitizer coupled amphiphilic macromolecular compound, a nanocarrier and a preparation method and application thereof, so as to solve the technical problems of poor biocompatibility, low bioavailability and weak targeting of antitumor drugs of photosensitizers in the prior art, resulting in poor antitumor effect.

[0007] To achieve the above purpose, the present application provides a hydroxyalkyl starch-photosensitizer coupled amphiphilic macromolecular compound, which is coupled by hydroxyalkyl starch and photosensitizer through a chemical bond; the photosensitizer is a hydrophobic porphyrin heterocyclic macrocyclic organic compound; the photosensitizer in the amphiphilic macromolecular compound serves as the hydrophobic end, and the hydroxyalkyl starch serves as the hydrophilic end.

[0008] Preferably, the hydrophobic porphyrin heterocyclic macrocyclic organic compound is one or more of mTHPC, chlorophyll-a, BPMppa, demethylated chlorophyllin a and pyro-demethylated chlorophyllin a.

[0009] Preferably, the hydroxyalkyl starch is hydroxymethyl starch, hydroxyethyl starch, hydroxypropyl starch or hydroxybutyl starch.

[0010] Preferably, the chemical bond is an amide bond or an ester bond.

[0011] According to another aspect of the present application, a preparation method of the amphiphilic macromolecular compound is provided, comprising the following steps: reacting a photosensitizer with a hydroxyalkyl starch in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine to generate a hydroxyalkyl starch-photosensitizer conjugate; wherein the molecular weight of the hydroxyalkyl starch is 25-480 kDa, and the molar substitution degree of the hydroxyalkyl group is 0.4-0.6.

[0012] According to another aspect of the present application, a nano-drug delivery system based on the amphiphilic macromolecular compound is provided, comprising the amphiphilic macromolecular compound, and further comprising a hydroxyalkyl starch-platinum (IV) conjugate.

[0013] Preferably, the nano-drug delivery system is prepared by emulsification, dialysis or high-pressure homogenization after mixing the hydroxyalkyl starch-photosensitizer amphiphilic macromolecular compound with the hydroxyalkyl starch-platinum (IV) conjugate.

[0014] Preferably, the mass ratio of the photosensitizer in the hydroxyalkyl starch-photosensitizer conjugate to the platinum element in the hydroxyalkyl starch-platinum (IV) conjugate is 1:(1-5), preferably 1:(1-2).

[0015] According to another aspect of the present application, the nano-drug delivery system is used in the preparation of a drug for treating and / or preventing cancer.

[0016] According to another aspect of the present application, an anticancer drug is provided, comprising the nano-drug delivery system and a pharmaceutically acceptable additive.

[0017] Overall, compared with the prior art, the above technical solutions conceived by the present application have the following beneficial effects:

[0018] (1) The hydroxyalkyl starch-photosensitizer conjugated amphiphilic macromolecular compound provided by the present application has a hydrophilic end of hydroxyalkyl starch and a hydrophobic end of a hydrophobic porphyrin heterocyclic macrocyclic organic compound. The hydroxyalkyl starch has good biocompatibility, biodegradability, non-toxicity, excellent water solubility and extremely low hypersensitivity. By connecting the hydroxyalkyl starch, the solubility of the photosensitizer is significantly improved, and its photodynamic performance is also retained.

[0019] (2) The photosensitizer used in the present application has a large planar molecular structure with large steric hindrance and rigidity, which cannot be directly connected to the main chain of some high molecular compounds. However, the hydroxyalkyl starch provides a connection site for the photosensitizer, so that the photosensitizer can be connected to the high molecular compound, solving the problem of poor solubility of the photosensitizer.

[0020] (3) The preparation method of the hydroxyalkyl starch-photosensitizer coupled amphiphilic macromolecular compound is simple, the condition is mild, the preparation method is easy to operate and control, and the safety is high.

[0021] (4) The hydrophobic porphyrin heterocyclic macrocyclic organic compound photosensitizer with photodynamic therapy characteristics is coupled with hydroxyalkyl starch as the hydrophobic end, and is assembled with hydroxyalkyl starch-platinum (IV) to form nanoparticles, and the preparation method is simple and easy to operate, the preparation condition is mild, and the cost is low. Experiments prove that the nanoparticles are prepared by mixing and co-assembling the photosensitizer-hydroxyalkyl starch macromolecular compound and hydroxyalkyl starch-platinum (IV), the reaction group of the photosensitizer and the hydroxyalkyl starch is not on the porphyrin ring of the photosensitizer, and after the co-assembly of the two, Pt does not appear in the center of the porphyrin ring of the photosensitizer in a coordinated manner, which may be the reason why the nano drug delivery system prepared by the application has a stable structure and a better tumor treatment effect. However, it is found in the experiment that when the photosensitizer and platinum (IV) are modified on the hydroxyalkyl starch to construct a nano drug delivery system, not only the drug uptake is very low, but also the tumor killing effect is very poor compared with the nano drug delivery system constructed by co-assembly.

[0022] (5) The hydrophobic porphyrin heterocyclic macrocyclic organic compound photosensitizer with photodynamic properties and certain selectivity to tumor cells is coupled with hydroxyalkyl starch as the hydrophobic end, and is assembled with hydroxyalkyl starch-platinum (IV) to form nanoparticles, and experiments find that the amphiphilic macromolecular compound can well improve the water solubility of the photosensitizer, and can also maintain the photodynamic properties of the photosensitizer, endow it with the characteristics of targeting tumors, improve the bioavailability of the photosensitizer, and the added hydroxyalkyl starch-platinum (IV) conjugate can release cisplatin in vivo, inhibit the activity of thioredoxin reductase, destroy the redox balance of tumors, promote the photodynamic therapy effect, and realize the targeted combination therapy and synergistic effect of anti-tumor drugs. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The flow chart for preparing the compound HP in Example 1 of the application;

[0024] Figure 2 The nuclear magnetic resonance hydrogen spectrum of hydroxyethyl starch, pyrocoll, and HP in Example 2 of the application;

[0025] Figure 3 The infrared spectrum of hydroxyethyl starch and HP in Example 2 of the application;

[0026] Figure 4 The hydration particle size distribution diagram of PtHP NP detected by a dynamic light scattering instrument in Example 3 of the application;

[0027] Figure 5The figure is a morphology diagram of PtHP NPs detected by an electron microscope in Embodiment 3 of the present application;

[0028] Figure 6 The figure is a stability diagram of PtHP NPs within 7 days detected by a dynamic light scattering instrument in Embodiment 3 of the present application;

[0029] Figure 7 The figure is a UV-Vis absorption spectrum of pyropheophorbide-a and PtHP NPs in Embodiment 4 of the present application;

[0030] Figure 8 The figure is a fluorescence emission spectrum of pyropheophorbide-a and PtHP NPs in Embodiment 4 of the present application;

[0031] Figure 9 The figure is a light stability of pyropheophorbide-a and PtHP NPs in vitro in Embodiment 5 of the present application;

[0032] Figure 10 The figure is a photodynamic performance of pyropheophorbide-a and PtHP NPs in vitro in Embodiment 5 of the present application;

[0033] Figure 11 The figure is a cellular uptake of HP and PtHP NPs by tumor cells detected by a laser confocal microscope in Embodiment 6 of the present application;

[0034] Figure 12 The figure is a cellular uptake of HP and PtHP NPs by tumor cells detected by a flow cytometer in Embodiment 6 of the present application;

[0035] Figure 13 The figure is an intracellular ROS generation induced by PtH, HP and PtHP NPs detected by a laser confocal microscope in Embodiment 7 of the present application;

[0036] Figure 14 The figure is an intracellular ROS generation induced by PtH, HP and PtHP NPs by tumor cells detected by a flow cytometer in Embodiment 7 of the present application;

[0037] Figure 15 The figure is a killing effect of HP and PtHP NPs on tumor cells under different drug concentrations in Embodiment 8 of the present application;

[0038] Figure 16 The figure is an influence of PtH and PtHP NPs on tumor cell TrxR in Embodiment 9 of the present application;

[0039] Figure 17 The figure is an influence of PtH and PtHP NPs on tumor cell GSH / GSSH ratio in Embodiment 9 of the present application;

[0040] Figure 18In vivo imaging images (content A) and relative quantitative detection images (content B) of PPa, HP and PtHP NPs in Example 10 of the present application for enrichment of the mouse tumor sites;

[0041] Figure 19 Fluorescence imaging images (content A) and relative quantitative detection images (content B) of PPa, HP and PtHP NPs in Example 10 of the present application for enrichment of each organ and tumor of the mouse;

[0042] Figure 20 The body weight change curve of the mouse after different administration treatments in Example 11 of the present application;

[0043] Figure 21 The tumor volume change curve of the mouse after different administration treatments in Example 11 of the present application;

[0044] Figure 22 The ex vivo tumor weight change graph of the mouse after different administration treatments in Example 11 of the present application;

[0045] Figure 23 The picture of the size of the stripped tumor of the mouse after different administration treatments in Example 11 of the present application;

[0046] Figure 24 The HE staining section graph of each organ of the mouse after different administration treatments in Example 11 of the present application;

[0047] Figure 25 The detection graph of the amount of white blood cells, red blood cells, hemoglobin and platelets in the blood of the mouse after different administration treatments in Example 11 of the present application;

[0048] Figure 26 The detection graph of the amount of creatinine, glutathione transaminase, glutathione transaminase, creatinine kinase and urea nitrogen in the serum of the mouse after different administration treatments in Example 11 of the present application;

[0049] Figure 27 The detection graph of the ROS generation in the tumor tissue of the mouse after different administration treatments in Example 11 of the present application;

[0050] Figure 28 The detection graph of the apoptosis in the tumor tissue of the mouse after different administration treatments in Example 11 of the present application;

[0051] Figure 29 The detection graph of the CD44 + CD24 - CSCs proportion in the tumor tissue of the mouse after different administration treatments in Example 11 of the present application;

[0052] Figure 30 Figure 11 is a detection chart of tumor infiltrating T cells in tumor tissues of mice treated with different administration methods in Example 11 of the present application;

[0053] Figure 31 Content (a) is a hydrated particle size distribution chart of platinum (IV) - hydroxyethyl starch- pyrolytic chlorophyllin-a nanometer particles (Pt-HES-PPa NP) prepared by Comparative Example 1, and content (b) is the stability of platinum (IV) - hydroxyethyl starch- pyrolytic chlorophyllin-a nanometer particles within 7 days detected by dynamic light scattering instrument;

[0054] Figure 32 Figure 9 is the fluorescence intensity of Pt-HES-PPa NP and HP and the control group detected by flow cytometry;

[0055] Figure 33 Figure 8 is the killing effect of HP and Pt-HES-PPa NP on tumor cells at different drug concentrations. DETAILED DESCRIPTION

[0056] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application.

[0057] The present application provides a kind of hydroxyalkyl starch-photosensitizer coupled amphiphilic macromolecular compound, the macromolecular compound is coupled by hydroxyalkyl starch and photosensitizer by chemical bond;The photosensitizer is hydrophobic porphyrin heterocyclic macrocyclic organic compound;The photosensitizer as hydrophobic end in the amphiphilic macromolecular compound, the hydroxyalkyl starch as hydrophilic end.

[0058] In some embodiments, the hydrophobic porphyrin-based heterocyclic macrocyclic organic compound is one or more of mTHPC (meso-tetra(4-hydroxyphenyl)chlorin), chlorophyll-a, BPMppa, methyl ester of monomethyl ether of pyropheophorbide-a, and pyropheophorbide-a. The hydroxyalkyl starch is hydroxymethyl starch, hydroxyethyl starch, hydroxypropyl starch, or hydroxybutyl starch. Preferably, the hydroxyalkyl starch is hydroxyethyl starch, the molecular weight of the hydroxyethyl starch is 25-480 kDa, the degree of substitution of the hydroxyethyl group is 0.4-0.6, and the specifications of the hydroxyethyl starch can be 480 / 0.4, 480 / 0.5, 480 / 0.6, 200 / 0.4, 200 / 0.5, 200 / 0.6, 130 / 0.4, 130 / 0.5, 130 / 0.6, 70 / 0.5, 25 / 0.5, preferably 130 / 0.4, wherein 130 represents the molecular weight of the hydroxyethyl starch in kDa, and 0.4 is the degree of substitution of the hydroxyethyl group. The chemical bond is an amide bond or an ester bond. In preferred embodiments, the photosensitizer is pyropheophorbide-a (PPa), and the pyropheophorbide-a is connected to the hydroxyalkyl starch through an ester bond.

[0059] The present application also provides a preparation method of the amphiphilic macromolecular compound, comprising the following steps: reacting a photosensitizer and a hydroxyalkyl starch in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine to generate a hydroxyalkyl starch-photosensitizer conjugate; wherein the molecular weight of the hydroxyalkyl starch is 25-480 kDa, and the molar degree of substitution of the hydroxyalkyl group is 0.4-0.6.

[0060] In some embodiments, the preparation method of the amphiphilic macromolecular compound comprises the following steps:

[0061] (1) mixing a photosensitizer dissolved in an organic solvent with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, and stirring for 0.4-0.6 hours to obtain a carboxyl-activated photosensitizer solution; the molar ratio of the photosensitizer to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:(3.5-4):(3.5-4); the concentration of the photosensitizer in the photosensitizer dissolved in the organic solvent is 3-5 mmol / L; and the organic solvent is dimethyl sulfoxide and / or N,N-dimethylformamide, etc.

[0062] (2) mixing the carboxyl-activated photosensitizer solution obtained in step (1) with a hydroxyalkyl starch, stirring at 25-40°C for 24-72 hours to allow esterification or amidation reaction to occur, after the reaction is completed, precipitating and washing the reaction product with isopropanol, centrifugally separating, dissolving the reaction product in an organic solvent, dialyzing, and freeze-drying to obtain the amphiphilic macromolecular compound. The mass ratio of the hydroxyalkyl starch to the photosensitizer is 4-6:1.

[0063] In some other embodiments, the hydroxyalkyl starch is modified with an amino group, and then an amidation reaction is carried out with a photosensitizer containing a carboxyl group, to obtain a hydroxyalkyl starch-photosensitizer amphiphilic macromolecular compound coupled by an amide bond.

[0064] In some embodiments, the drug loading of the photosensitizer in the amphiphilic macromolecular compound is 1-5 wt%, more preferably 2-4 wt%.

[0065] The present application also provides a nano-drug delivery system based on the amphiphilic macromolecular compound, which comprises the amphiphilic macromolecular compound and a hydroxyalkyl starch-platinum(IV) conjugate.

[0066] In some embodiments, the nano-drug delivery system is prepared by emulsification, dialysis or high-pressure homogenization after mixing the hydroxyalkyl starch-photosensitizer amphiphilic macromolecular compound with the hydroxyalkyl starch-platinum(IV) conjugate.

[0067] In some embodiments of the present application, the hydroxyalkyl starch-platinum(IV) conjugate is prepared by the following method:

[0068] (1) Pt(IV)-(OH)2 is generated by reacting cisplatin with hydrogen peroxide at 45-60°C;

[0069] (2) Pt(IV)-(COOH)x, 1.0≤x<2.0, an axially carboxylate-coordinated platinum(IV) complex, is generated by reacting Pt(IV)-(OH)2 obtained in step (1) with succinic anhydride;

[0070] (3) A hydroxyethyl starch-platinum(IV) conjugate is generated by reacting the axially carboxylate-coordinated platinum(IV) complex Pt(IV)-(COOH)x, 1.0≤x<2.0, obtained in step (2) with hydroxyethyl starch in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP).

[0071] The mass ratio of the photosensitizer in the hydroxyalkyl starch-photosensitizer conjugate to the platinum element in the hydroxyalkyl starch-platinum(IV) conjugate is 1:(1-5), more preferably 1:(1-2).

[0072] In some embodiments, the preparation method of the nano-drug delivery system comprises the following steps: mixing the hydroxyalkyl starch-photosensitizer coupled amphiphilic macromolecular compound and the hydroxyalkyl starch-platinum (IV) conjugate according to a mass ratio of 1:0.8-1.2, dissolving them in ultrapure water, then adding an organic solvent, wherein the volume ratio of the ultrapure water to the organic solvent is (1-3 mL):(100-300 μL), then performing ultrasonic emulsification, removing the organic solvent from the emulsified solution, and finally performing constant volume with ultrapure water to obtain the nano-drug delivery system.

[0073] In some embodiments, the hydroxyalkyl starch-photosensitizer coupled amphiphilic macromolecular compound and the hydroxyalkyl starch-platinum (IV) conjugate are mixed according to a mass ratio of 1:0.8-1.2, and then dissolved in ultrapure water, wherein the mass ratio of the hydroxyalkyl starch-photosensitizer coupled amphiphilic macromolecular compound to the ultrapure water is 1:1.8-2.2. The organic solvent is dichloromethane, dichloroethane, trichloromethane, etc., the ultrasonic power is 120-180 W, the ultrasonic time is 1.5-3 min, the working time is 1-3 s, and the intermittent time is 1-3 s.

[0074] In some embodiments, the drug loading amount of the photosensitizer in the nano-drug delivery system is 0.5-2.5%.

[0075] The nano-drug delivery system of the present application can be used for preparing a drug for treating and / or preventing cancer, and therefore the present application further provides an anticancer drug comprising the nano-drug delivery system and a pharmaceutically acceptable additive. The anticancer drug of the present application is suitable for treating cancer tumors including but not limited to breast cancer, liver cancer, colon cancer, ovarian cancer or melanoma, etc. The dosage form of the anticancer drug can be injection, powder injection, oral dosage, spray, capsule or suppository.

[0076] The hydrophilic end of the hydroxyalkyl starch-photosensitizer coupled amphiphilic macromolecular compound provided by the present application is hydroxyalkyl starch, and the hydrophobic end is a hydrophobic porphyrin heterocyclic macrocyclic organic compound. The present application uses a hydrophobic porphyrin heterocyclic macrocyclic organic compound photosensitizer having a photodynamic property and a certain selectivity for tumor cells as the hydrophobic end, which is coupled with hydroxyalkyl starch and assembled into nanoparticles with hydroxyalkyl starch-platinum (IV) conjugate. It has been found that the amphiphilic macromolecular compound can well improve the water solubility of the photosensitizer, and also can maintain the photodynamic property of the photosensitizer, endow it with the characteristics of targeting tumor, improve the bioavailability of the photosensitizer, and the added hydroxyalkyl starch-platinum (IV) conjugate can release cisplatin in vivo to inhibit the activity of thioredoxin reductase, destroy the redox balance of tumor, promote the effect of photodynamic therapy, and the specific co-assembly mode realizes the targeted and synergistic combination therapy of photosensitizer and cisplatin drugs.

[0077] The following are specific embodiments:

[0078] Example 1

[0079] The present invention provides a hydroxyalkyl starch-photosensitizer amphiphilic macromolecular compound, which is obtained by chemically coupling hydroxyalkyl starch and a photosensitizer. The hydroxyalkyl starch is hydroxyethyl starch 130 / 0.4, and the photosensitizer is pyropheophorbide-a, and the two are coupled by an ester bond. The synthetic route is as follows: Figure 1 The specific operations are as follows:

[0080] (1) 40 mg of pyropheophorbide-a (PPa, 0.077 mmol) was dissolved in 20 mL of dimethyl sulfoxide, 59 mg (0.3 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (ECCI) and 37.5 mg (0.3 mmol) of 4-dimethylaminopyridine (DMAP) were added as catalysts, and the reaction was stirred at room temperature for 0.5 h to activate the carboxyl group;

[0081] (2) Add 200 mg of hydroxyethyl starch (HES, 130 / 0.4) to the solution described in (1) and stir in an oil bath at 30°C for 48 h;

[0082] (3) After the reaction as described in (2) is completed, the reaction product is precipitated with 10 volumes of isopropanol and collected by centrifugation, and then the reaction product is washed three times with isopropanol; the centrifugation speed is 9000 rpm, and the centrifugation time is 5 min;

[0083] (4) After washing, the product was dissolved in dimethyl sulfoxide and dialyzed with ultrapure water for 3 days, and then freeze-dried to obtain a dark green solid; the dark green solid was the target product, hydroxyethyl starch-pyropheophorbide-a conjugate (HES-PPa, HP).

[0084] Example 2

[0085] Confirmation of the structure of hydroxyethyl starch-pyropheophorbide-a conjugate (HES-PPa, HP): The chemical structure of the hydroxyethyl starch-pyropheophorbide-a conjugate prepared in Example 1 was confirmed by H NMR and FTIR. Figure 2 and Figure 3 The H NMR spectrum and FTIR spectrum of the hydroxyethyl starch-pyropheophorbide-a conjugate prepared in Example 1 are shown in FIG. Figure 2) shows that, compared to hydroxyethyl starch, after coupling with pyropheophorbide-a, the H NMR spectrum of the hydroxyethyl starch-pyropheophorbide-a conjugate exhibits multiple proton peaks between 6 and 9 ppm, which are attributed to the porphyrin ring of pyropheophorbide-a (δ = 9.74, 9.47, 8.91, 8.24, 6.41, and 6.23 ppm, respectively. The appearance of these new proton peaks indicates that the hydroxyethyl starch-pyropheophorbide-a conjugate was successfully synthesized.

[0086] From the infrared spectrum ( Figure 3 ) It can be seen that compared with hydroxyethyl starch, the infrared absorption of hydroxyethyl starch-pyropheophorbide-a conjugate is at 1728cm -1 and 1233cm -1 New absorption peaks appeared at 100 nm, attributable to the C=O stretching vibration and the C-O-C stretching vibration of the ester bond generated by the reaction, respectively. This result indicates that pyropheophorbide-a is coupled to hydroxyethyl starch via an ester bond, forming a hydroxyethyl starch-pyropheophorbide-a conjugate. Proton nuclear magnetic resonance (HNMR) and infrared (IR) spectroscopy confirmed the correct chemical structure of the hydroxyethyl starch-pyropheophorbide-a conjugate prepared in Example 1. UV spectrophotometry confirmed the drug loading to be 3.5%.

[0087] Example 3

[0088] Preparation and characterization of nanodrug delivery system (PtHP NP).

[0089] First, a hydroxyalkyl starch-platinum (IV) conjugate was prepared by the following method:

[0090] (1) Weigh 1 g of cisplatin (3.33 mmol) and place it in a 100 mL round-bottom flask. Then, add 35 mL of 30% hydrogen peroxide (10-fold excess) and 25 mL of ultrapure water to the flask in sequence. Reflux at 50°C for 1 h. After the reaction is complete, place in an ice bath and recrystallize the product. Collect the supernatant by centrifugation and wash three times with ethanol and ether. After vacuum drying, the resulting white solid powder is the cisplatin oxidation product, Pt(IV)-(OH)2.

[0091] (2) The obtained Pt(IV)-(OH)2500 mg (1.5 mmol) was dispersed in 40 mL of dimethyl sulfoxide, and 150.1 mg (1.5 mmol) of succinic anhydride was added, and the reaction was stirred at 45 °C for 12 h. The obtained reaction solution was poured into a mixed solution of diethyl ether / ethanol, stirred, centrifuged at 9000 rpm for 5 min, and a white precipitate was obtained. The white precipitate was washed with acetone and diethyl ether, respectively, and then dried under vacuum at room temperature to obtain a dried white solid as a platinum(IV) complex coordinated with axial carboxylic acid, Pt(IV)-(COOH)x;

[0092] (3) The obtained cis-dichlorohydroxy diammine platinum(IV) succinate monoester 100 mg (0.23 mmol) was dispersed in 16 mL of N,N-dimethylformamide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride 220.1 mg (1.15 mmol) and 4-dimethylaminopyridine 71.0 mg (0.58 mmol) were added as catalysts, and the reaction was stirred at room temperature for 0.5 h. Then, hydroxyethyl starch 100 mg was added, and the reaction was stirred at 45 °C for 48 h. The obtained reaction solution was poured into isopropanol to precipitate the reaction product, which was collected by centrifugation. The precipitate was washed with isopropanol, then dissolved in DMSO, and dialyzed against ultrapure water for 3 days. After freeze-drying, a solid was obtained as a hydroxyethyl starch-platinum(IV) conjugate, wherein the molecular weight cut-off of the dialysis bag was 3500 Da.

[0093] The preparation method of the nano drug delivery system (PtHP NP) nanoparticles is as follows:

[0094] (1) 1 mg of hydroxyethyl starch-pyropheophorbide-a conjugate and 1 mg of hydroxyalkyl starch-platinum(IV) (HES-Pt, PtH) were dissolved in 2 mL of water, and the total mass of the two samples was 2 mg. The mass ratio of pyropheophorbide-a to platinum was 1:2.

[0095] (2) The above sample was dissolved in 2 mL of ultrapure water, and 200 μL of dichloromethane was added to the solution. Emulsification was performed by a cell sonicator (ultrasonic power 150 W, ultrasonic time 2 min, working time 2 s, and intermittent time 2 s).

[0096] (3) After ultrasonic emulsification, the emulsified solution was removed by rotary evaporation (37 °C, 4 min), and then the solution was diluted to 2 mL with ultrapure water to obtain the target nanoparticles (PtHP NP).

[0097] Morphological analysis:

[0098] The particle size of PtHP NP was detected by a laser particle size analyzer, and the particle size detection results were as follows: Figure 4The results show that uniform PtHP NPs were prepared with a particle size of 177.8±2.8nm and a PDI of 0.145. PtHP NPs were diluted 50 times and 10μL was dropped on a copper grid. After natural drying at room temperature, the morphology was observed using a transmission electron microscope. Figure 5 It can be seen that the particle size of PtHP NP is about 170nm. At the same time, the nanoparticles maintain good stability in aqueous solution, and no obvious agglomeration or disaggregation occurs for a week ( Figure 6 ).

[0099] Comparative Example 1

[0100] 200 mg of hydroxyethyl starch-pyropheophorbide-a conjugate was reacted with 200 mg of axially carboxylic acid-coordinated platinum (IV) complex Pt(IV)-(COOH)x, 1.0≤x<2.0, in the presence of 440.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 142 mg of 4-dimethylaminopyridine (DMAP) at 30°C for 48 h to obtain platinum (IV)-hydroxyethyl starch-pyropheophorbide-a conjugate (Pt-HES-PPa), wherein the drug loading of PPa was 3% and the drug loading of Pt was 1%, which were lower than the drug loading in the nanoparticles in Example 3. The possible reason is that PPa and Pt(IV)-(COOH)x have the same reaction sites as hydroxyethyl starch, and the two compete for the reaction sites on HES, resulting in a low drug loading of the subsequent Pt(IV)-(COOH)x.

[0101] 2 mg of Pt-HES-PPa was weighed and dissolved in 2 mL of aqueous solution. 200 μL of dichloromethane was added to the solution, and emulsification was performed using a cell ultrasonic disruptor (ultrasonic power 150 W, ultrasonic time 2 min, working time 2 s, rest time 2 s). After ultrasonic emulsification, the emulsified solution was subjected to rotary evaporation to remove the dichloromethane (37°C, 4 min), and the solution was then diluted to 2 mL with ultrapure water to obtain platinum (IV)-hydroxyethyl starch-pyropheophorbide-a nanoparticles (Pt-HES-PPa NPs).

[0102] like Figure 31 As shown in Figures (a) and (b), the Pt-HES-PPa NPs had a particle size of 168.8 nm and a PDI of 0.218. The nanoparticles also maintained excellent stability in aqueous solution, showing no significant aggregation or disaggregation over a week.

[0103] Although the particle size and stability of Pt-HES-PPa NPs are similar to those of PtHP NPs, their Pt loading is relatively low and the ratio of the two cannot be adjusted, resulting in poor practicality.

[0104] Example 4

[0105] UV-Vis absorption spectra and fluorescence emission spectra of PtHP NPs. The mixed solution of ultrapure water and dimethyl sulfoxide was used to dilute PtHP NPs and PPa. The absorption spectra of samples were detected using a UV spectrophotometer with the mixed solution of ultrapure water and dimethyl sulfoxide as a reference, and the wavelength scanning range was 400-800 nm with a scanning wavelength of 1 nm. The results are shown in Figure 7 The results show that the Soret band and Q band of the porphyrin ring of PPa do not change significantly after modification and assembly, indicating that the reaction groups of PPa and HES are not on the porphyrin ring, and will not have too much impact on the function of PPa. In addition, the four peaks of the Q band do not change significantly after HES-Pt assembly, indicating that Pt does not appear in the center of the porphyrin ring in a coordinated manner.

[0106] The mixed solution of ultrapure water and dimethyl sulfoxide was used to dilute PtHP NPs and PPa. The fluorescence spectra of samples were detected using a fluorescence spectrometer. The excitation wavelength was 488 nm, and the scanning wavelength was 620-800 nm. The results are shown in Figure 8 The results show that compared with free PPa, the peak shape of the fluorescence spectrum of PtHP NPs does not change significantly, but the fluorescence intensity is slightly weakened, and the possible reason is that the light is quenched by aggregation. Free PPa is in a dispersed state, and after modification and assembly, PPa is largely aggregated on the HES molecule, resulting in the occurrence of light quenching by aggregation.

[0107] Example 5

[0108] Light stability and photodynamic performance detection of PtHP NPs. In order to explore the light stability of PtHP NPs, PtHP NPs and PPa were dissolved in DMSO to make the concentration of PPa 10 μg / ml, 1 mL was taken and added to a 48-well plate, a 660 nm laser with a power of 100 mW / cm 2 was used for irradiation, and the absorbance value at 668 nm was detected at 0, 5, 10, 20, 30, 60, 90, 120, 150, and 180 s, respectively. The results are shown in Figure 9 Free PPa will decompose under light and its concentration will gradually decrease, losing activity; after PPa is modified by HES, its light stability is enhanced, indicating that the modification of PPa by HES can solve the problem of poor light stability caused by the aggregation of PPa in aqueous solution. The results show that the light stability of PtHP NPs is significantly improved compared with free PPa.

[0109] To investigate the photodynamic performance of PtHP NPs, 1 mg of 1,3-diphenyl isobenzofuran (DPBF) was dissolved in 1 mL of ethanol to obtain a DPBF stock solution (1 mg / mL). Then 25 μΐ of the DPBF solution was added to 1 mL of PPa, PtHP NPs or DMSO solution without PPa (PPa 1 μg / mL), and 1 mL of the corresponding solution was added to a 48-well plate. Then the mixture was irradiated using a 660 nm laser at 200 mW / cm2, and the absorbance at 405 nm was detected every 5 seconds. The decay of the absorbance of DPBF at 405 nm over time represents the ROS generation ability. The results are shown in 2 Fig. 6, which show that PtHP NPs and PPa have essentially the same photodynamic effect, indicating that the modification and assembly of PPa do not reduce the photodynamic effect of PPa. Figure 10

[0110] Example 6

[0111] Investigation of the cellular uptake of PtHP NPs. First, the uptake of nanomaterials by tumor cells was qualitatively investigated by confocal microscopy. The cultured 4T1 cells were diluted with 1640 complete medium and then plated in confocal dishes, with 150,000 cells per dish. The cells were incubated in an incubator for 12 h to allow them to grow adherently. After the cells adhered, the original culture medium was aspirated, and 1 mL of PtHP NP and HP solution diluted with culture medium to a final concentration of 1 μg / mL of PPa was added. The control group did not contain drugs. The cells were incubated in an incubator for 12 h. After 12 h of incubation, the original culture medium was aspirated, and 1 mL of 10 μg / mL DAPI was added for staining. After 15 min of staining, the DAPI was aspirated, and PBS was used for washing. After three washes, the fluorescence of PPa in 4T1 cells was detected using a laser confocal microscope, and the results are shown in Figure 11 Fig. 7. The results show that both PtHP NPs and HP can be well taken up by tumor cells at the cellular level.

[0112] ​After the qualitative analysis by confocal microscope, the uptake of the NPs by the tumor cells was quantitatively investigated by flow cytometry. The cultured 4T1 cells were diluted with 1640 complete medium and then plated into 6-well plates at 200,000 cells per well, and incubated in an incubator for 12 h to allow them to grow adherently. After the cells adhered, the original culture medium was aspirated, and 2 ml of PtHP NPs and HP solution diluted with the culture medium to a final concentration of 1.5 μg / ml of PPa was added, and a group without drug was used as a control, and incubated in an incubator for 12 h. After 12 h of incubation, the original culture medium was aspirated, and washed with PBS three times, and then the cells in each well were trypsinized for 3 min with 300 μL of trypsin. After trypsinization was completed, 700 μl of 1640 complete medium was added to terminate the trypsinization, and the cells were collected by centrifugation at 1500 rpm for 3 min. The collected cells were washed with PBS three times, and then resuspended with 200 μL of PBS. The fluorescence intensity of PPa was detected by flow cytometry, and the results are shown in Figure 12 The results show that PtHP NPs and HP can be well taken up by tumor cells at the cell level, and the results are consistent with those of confocal.

[0113] Comparative Example 2

[0114] Investigation of Pt-HES-PPa NP cell uptake. The cultured 4T1 cells were diluted with 1640 complete medium and then plated into 6-well plates at 200,000 cells per well, and incubated in an incubator for 12 h to allow them to grow adherently. After the cells adhered, the original culture medium was aspirated, and 2 ml of Pt-HES-PPa NPs and HP solution diluted with the culture medium to a final concentration of 1.5 μg / ml of PPa was added, and a group without drug was used as a control, and incubated in an incubator for 12 h. After 12 h of incubation, the original culture medium was aspirated, and washed with PBS three times, and then the cells in each well were trypsinized for 3 min with 300 μL of trypsin. After trypsinization was completed, 700 μl of 1640 complete medium was added to terminate the trypsinization, and the cells were collected by centrifugation at 1500 rpm for 3 min. The collected cells were washed with PBS three times, and then resuspended with 200 μL of PBS. The fluorescence intensity of PPa was detected by flow cytometry, and the results are shown in Figure 32 The results show that the uptake of Pt-HES-PPa NPs by tumor cells is significantly weaker than that of HP, indicating that the design of Pt-HES-PPa NPs has the defect of reducing drug uptake, further indicating the necessity of preparing PtHP NPs.

[0115] Example 7

[0116] ROS production ability of PtHP NPs under light was explored. Intracellular ROS was detected by using fluorescent probe DCFH-DA. First, the uptake of nanometer particles by tumor cells was qualitatively explored by confocal microscopy. The cultured 4T1 cells were diluted with 1640 complete medium and then plated into confocal dishes, 150,000 cells per dish, and incubated in an incubator for 12 h to allow them to grow adherently. After the cells adhered, the original culture medium was aspirated, 1 ml of PtHP NP and HP solution diluted with culture medium to a final concentration of 0.5 μg / mL was added, and the group without drug was used as a control, and incubated in an incubator for 12 h. After 12 h of incubation, the original culture medium was aspirated, and 1 mL of 10 μmol / mL DCFH-DA culture medium was added for incubation. After 30 min of incubation, the DCFH-DA-containing medium was aspirated, and PBS was used for washing, which was repeated three times. After washing, PBS was used for incubation, and a 660 nm laser was used for laser irradiation, 40 s per dish, and the light power was 10 mW / cm 2 The fluorescence of DCFH-DA in 4T1 cells was detected by laser confocal microscopy, and the results are shown in Figure 13 . The results show that PtH can slightly increase the ROS level in 4T1 cells, HP has a strong photodynamic effect and can significantly increase the ROS level in 4T1 cells, and the ROS level in PtHP NP group is the highest, and the possible reason is that PtHP NPs can release cisplatin, affect the redox balance in cells, and enhance the ROS burst growth caused by photodynamic therapy.

[0117] After qualitative analysis by confocal microscopy, the uptake of nanometer particles by tumor cells was quantitatively explored by flow cytometry. The cultured 4T1 cells were diluted with 1640 complete medium and then plated into 6-well plates, 150,000 cells per well, and incubated in an incubator for 12 h to allow them to grow adherently. After the cells adhered, the original culture medium was aspirated, 2 mL of PtHP NP and HP solution diluted with culture medium to a final concentration of 0.5 μg / mL was added, and the group without drug was used as a control, and incubated in an incubator for 12 h. After 12 h of incubation, the original culture medium was aspirated, and 1 mL of 10 μmol / mL DCFH-DA culture medium was added for incubation. After 30 min of incubation, the DCFH-DA-containing medium was aspirated, and PBS was used for washing, which was repeated three times. After washing, PBS was used for incubation, and a 660 nm laser was used for laser irradiation, 40 s per well, and the light power was 10 mW / cm 2 The fluorescence of DCFH-DA in 4T1 cells was detected by laser confocal microscopy, and the results are shown in Figure 14 . The results show that the ROS level in 4T1 cells in the PtHP NP group is significantly higher than that in the HP and PtH groups, indicating that PtHP NPs have the best ability to induce the generation of intracellular ROS.

[0118] Example 8

[0119] Evaluation of the anti-tumor activity of PtHP NPs in vitro. The cultured 4T1 cells were diluted with 1640 complete medium and then plated into 96-well plates at 5000 cells per well, and incubated in an incubator for 12 h to allow them to adhere. The medium containing PtHP NPs and HP was prepared in a concentration gradient of 5, 1.25, 0.32, 0.08 and 0.02 μg / mL of PPa. After the cells adhered, the original culture medium was aspirated, and the prepared medium containing different concentrations of PtHP NPs and HP was added for incubation at 200 μL per well, with 3 wells per group, and blank and cell-free control groups were set. After 24 h of incubation, laser irradiation was performed using a 660 nm laser with a power of 10 mW / cm 2 , for 20 s per well. After the irradiation was completed, the 96-well plates were placed in an incubator for 24 h. After the incubation was completed, the original culture medium was aspirated, and culture medium containing 10% CCK-8 was added at 100 μL, and incubated for about 1 h, after which the OD value at 450 nm of each well of the 96-well plate was detected using a microplate reader, and the results are shown in Figure 15 . The IC 50 of PtHP NPs and HP calculated by Graphpad was 0.19 μg / mL and 0.42 μg / mL, respectively. The IC 50 of PtH was obtained as 2.28 μg / mL (mass of Pt) by the same method. According to the IC 50 values obtained from each experimental group, the combination index (CI) was calculated according to the following formula:

[0120]

[0121] (D)1: the drug concentration at which 50% inhibition is calculated for drug 1 in combination;

[0122] (D)2: the drug concentration at which 50% inhibition is calculated for drug 2 in combination;

[0123] (D 50 )1: the drug concentration at which 50% inhibition is calculated for drug 1 in combination;

[0124] (D 50 )2: the drug concentration at which 50% inhibition is calculated for drug 2 in combination.

[0125] The CI of PtHP NPs was calculated to be 0.6124 < 1, indicating that there is good synergy in the joint action of the two components. The amount of ROS generated has a certain relationship with cytotoxicity, and this result is consistent with the fact that PtHP NPs have the best ability to induce the generation of intracellular ROS in Example 10.

[0126] Comparative Example 3

[0127] Evaluation of in vitro anti-tumor activity of Pt-HES-PPa NPs.

[0128] The cultured 4T1 cells were diluted with 1640 complete medium and then plated into 96-well plates at 5000 cells per well, and incubated in an incubator for 12 h to allow them to adhere. The medium containing Pt-HES-PPa NPs and HP was prepared in a concentration gradient of PPa of 0.5, 0.25, 0.125, and 0.0625 pg / mL. After the cells adhered, the original culture medium was removed, and the prepared medium containing different concentrations of Pt-HES-PPa NPs and HP was added for incubation at 200 pL per well, with 3 wells per group, and blank and cell-free control groups were set. After 24 h of incubation, laser irradiation was performed using a 660 nm laser at a power of 10 mW / cm 2 for 20 s per well. After the irradiation was completed, the 96-well plate was placed in the incubator for 24 h. After the incubation was completed, the original culture medium was removed, and culture medium containing 10% CCK-8 was added at 100 pL, and incubated for about 1 h, followed by detection of the OD value at 450 nm for each well of the 96-well plate using a microplate reader. The results are shown in Figure 33 , which show that the cell-killing effect of Pt-HES-PPa NPs is weaker than that of HP. In combination with Figure 14 , it can be seen that the cell-killing effect of Pt-HES-PPa NPs is significantly weaker than that of PtHP NPs and the uptake of Pt-HES-PPa NPs by tumor cells is weaker than that of HP, which is consistent with the results of Comparative Example 2. In summary, Comparative Examples 1, 2, and 3, although the particle size and stability of Pt-HES-PPa NPs are similar to those of PtHP NPs, there are problems such as low drug loading, difficult to control the ratio, poor uptake by cells, etc.

[0129] Example 9

[0130] To explore the way PtHP NPs destroy the redox balance. Thioredoxin reductase (TrxR) and thioredoxin (Trx), NADPH together constitute the thioredoxin system. It has been reported that cisplatin can inhibit the activity of TrxR, and the reduction of oxidized Trx is mediated by TrxR and NADPH system. PtHP NPs can release cisplatin, which may have an inhibitory effect on TrxR, which was verified by a TrxR kit. The cultured 4T1 cells were diluted with 1640 complete medium and then plated into 6-well plates at 300,000 cells per well, and incubated in an incubator for 12 h to allow them to adhere. After the cells adhered, the original culture medium was removed, and PtHP NPs and PtH solution diluted with culture medium to a final concentration of Pt of 2 pg / mL were added at 2 mL, and a drug-free group was used as a control, and incubated in an incubator for 12 h. After 12 h of incubation, the subsequent experiments were performed according to the instructions of the thioredoxin reductase (TrxR) activity assay kit (Solabio). The experimental results are as follows:Figure 16 The results show that PtH has the ability to inhibit the activity of TrxR, and PtHPNP has a better inhibitory effect.

[0131] In most eukaryotes, there are two independent antioxidant systems, one is the thioredoxin system, and the other is the glutathione system. When the thioredoxin system is inhibited, the glutathione system will also be affected to a certain extent. The GSH / GSSG ratio can be used to reflect the intracellular redox state. The effect of PtHPNP on intracellular redox level was explored by detecting the GSH / GSSG ratio. The cultured 4T1 cells were diluted with 1640 complete medium and then plated into a 6-well plate, 300,000 cells per well, and incubated in an incubator for 12 h to allow them to grow adherently. After the cells adhered, the original culture medium was aspirated, and 2 mL of PtHPNP and PtH solution diluted with culture medium to a final concentration of 2 μg / mL of Pt were added, and the drug-free group was used as a control, and incubated in an incubator for 12 h. After 12 h of incubation, the intracellular GSH / GSSG ratio was measured using a GSH and GSSG detection kit (Bi Yun Tian), and the results are shown in Figure 17 The results show that PtH and PtHPNP can both destroy the intracellular redox balance. The possible reason for this result is that the PtH component can release cisplatin in the cell, thereby destroying the intracellular redox balance.

[0132] Example 10

[0133] PtHPNP in vivo tissue distribution study. 6-week-old BALB / c female mice were purchased, with a body weight of about 19 g. After adaptive feeding in the laboratory animal room for one week, the hair around the right hind limb of the mice was shaved clean. While feeding the mice, 4T1 cells were cultured for subcutaneous tumor inoculation. At the time of inoculation, 4T1 cells were resuspended to a cell suspension of 10 7 / mL. A syringe was used to subcutaneously inject 100 μL of cell suspension above the right hind limb of each mouse. After injection, continue to feed. The tumor volume calculation formula is: V = (L x W 2 ) / 2, where V represents the tumor volume, L represents the long diameter of the tumor, and W represents the short diameter. When the tumor volume reaches 200 mm 3 , the tumor-bearing mice were randomly divided into 3 groups, 3 mice in each group. Free PPa, HP and PtHPNP were injected into the tail vein respectively, and the dose was calculated as 3 mg / kg of PPa. Before and after administration at 1, 2, 4, 8, 12, and 24 h, the mice were anesthetized, and the mice were imaged using a small animal imaging instrument. The imaging results and quantitative results are shown in Figure 18Content A and Content B. The results show that free PPa is quickly eliminated in the tumor, while HP has a longer residence time but a lower enrichment concentration, and the PtHP NP group shows the highest enrichment concentration and longer residence time. This can further enhance the therapeutic effect of photodynamic therapy, and the long residence time in the tumor makes the choice of irradiation time more flexible during treatment, and the real-time changes of the tumor can also be monitored by detecting the fluorescence signal during treatment.

[0134] To further study the distribution behavior of PtHP NP in vivo, the mice were sacrificed 24 h after administration, and the heart, liver, spleen, lung, kidney and tumor were removed. The fluorescence images of each tissue and tumor were collected using a small animal imaging instrument, and the fluorescence was quantitatively analyzed. The results are shown in Figure 19 Content A and Content B. The results show that free PPa is quickly eliminated in the tumor, while HP has a longer residence time but a lower enrichment concentration, and the PtHP NP group shows the highest enrichment concentration and longer residence time. This can further enhance the therapeutic effect of photodynamic therapy, and the long residence time in the tumor makes the choice of irradiation time more flexible during treatment, and the real-time changes of the tumor can also be monitored by detecting the fluorescence signal during treatment.

[0135] Example 11

[0136] In vivo anti-tumor evaluation of PtHP NP. BALB / c female mice, six weeks old, weighing about 19 g, were purchased. After adaptive feeding in the laboratory animal room for one week, the hair around the right hind limb of the mice was shaved clean. While feeding the mice, 4T1 cells were cultured for subcutaneous tumor inoculation. At the time of inoculation, 4T1 cells were resuspended to a cell suspension of 10 7 / mL. A syringe was used to subcutaneously inject 100 μL of cell suspension on the right hind limb of each mouse. After injection, continue to feed. The tumor volume calculation formula is: V = (L x W 2 ) / 2, where V represents the tumor volume, L represents the long diameter of the tumor, and W represents the short diameter. When the tumor volume reaches 120 mm 3 , it is recorded as the first day. The tumor-bearing mice were randomly divided into 6 groups, 8 mice in each group. Different drugs were injected into the tail vein. G1: normal saline; G2: Cisplatin + PPa + Laser; G3: PtH; G4: PtHP; G5: HP + Laser; G6: PtHP + Laser (L in the figure represents Laser, and Cisplatin represents cisplatin). The dose of the drug is PPa 1 mg / kg, Pt 2 mg / kg. The drug is given every three days, a total of two times, and 660 nm laser is used for irradiation 12 h after each administration. The light irradiation power is 600 mW / cm 2, light time was 5 min. During the experiment, the weight of mice and tumor volume were measured every other day. The results are shown in Figure 20 , Figure 21 , The mice were sacrificed on the 15th day, the tumors were peeled off, and the ex vivo tumors were weighed and photographed. The results are shown in Figure 22 , Figure 23 , At the same time, three hearts, livers, spleens, lungs, and kidneys were taken from each group, respectively, fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned for HE staining. The results are shown in Figure 24 , After the mice were sacrificed, two portions of whole blood were collected from each mouse. One portion was used to determine the blood routine (anticoagulant) index, and the other portion was used to detect the blood biochemical (non-anticoagulant) index. The whole blood for blood routine determination can be directly detected by a blood cell analyzer, and the whole blood for blood biochemical index determination needs to be centrifuged at 3000 rpm for 5 min after being placed at 4°C overnight. The serum was collected and then detected using the corresponding kit. The results are shown in Figure 25 , Figure 26 ,

[0137] The remaining tumor was prepared into a single cell suspension: the peeled tumor was washed with PBS and placed in a 1.5 mL EP tube. It was chopped with sterile ophthalmic scissors and 1 mL of digestion solution containing collagenase IV (Biosharp) and DNase I (Biosharp) was added. It was digested for 1 h, shaking every 30 min. After digestion was complete, the cells were placed on a sieve and serum-containing medium was added. The cells were squeezed vertically downward with a sterile syringe rubber head, and the suspension was filtered through a 200-mesh filter. Centrifugation was performed at 2000 rpm, 4°C, for 5 min. PBS was added for washing, and centrifugation was performed at 2000 rpm, 4°C, for 5 min. PBS 2 mL was added to resuspend the cells, and 0.5 mL was used to detect ROS, 0.5 mL was used to detect apoptosis, and 0.5 mL was used to detect tumor stem cells (CSCs). G1 and G6 were taken, and 0.5 mL was used to detect tumor infiltrating T cells.

[0138] ROS detection in tumor tissue: DCFH-DA probe was used to stain the single cell suspension of tumor tissue in different groups, and flow cytometry was used for analysis. The results are shown in Figure 27 .

[0139] Detection of apoptotic cells in tumor tissue: after obtaining the single cell suspension, the cells were washed twice with PBS, suspended in 300 μL PBS, stained with ANNEXIN V-FITC / PI apoptosis detection kit, and the apoptosis of the cells was detected by flow cytometry. The results are shown in Figure 28 .

[0140] Detection of CSCs in tumor tissue: after obtaining the single cell suspension, the cells were washed twice with PBS, suspended in 300 μL PBS, and stained with CD44, CD24 antibodies. The CSCs were detected by CD44+ CD24 - The biomarker method was used to identify the proportion of CSCs in tumor tissues. The results are as follows Figure 29 shown.

[0141] Detection of tumor-infiltrating T cells: After obtaining a single cell suspension, wash the cells twice with PBS, suspend them in 300 μL PBS, separate the lymphocytes using Ficoll, and stain them with CD3, CD4, and CD8 antibodies. Detect and analyze them on a flow cytometer. The results are as follows: Figure 30 shown.

[0142] Figure 20 Body weight data showed that PtHP NP had good safety and no obvious toxic side effects. Figure 21 Tumor volume data showed that HP had a better therapeutic effect, and PtHP NP had the best therapeutic effect, which may be related to its longer retention time and stronger tumor targeting and enrichment ability. In addition to tumor volume, tumor weight and tumor photos can also reflect the therapeutic effect. Figure 22 Tumor weight showed that PtHP NP had the best therapeutic effect. Figure 23 The tumor photos showed that PtHP NP had the best therapeutic effect. The damage of different drugs to various organs of mice was evaluated by HE staining sections. Figure 24 The results showed that PtHPNP did not cause significant damage to various organs and had no significant toxicity. Blood routine and blood biochemistry can also reflect the safety of the drug to a certain extent. Figure 25 ,26 The results of blood routine and blood biochemistry also showed that PtHP NP has good safety and no obvious toxic side effects. Photosensitizers kill tumor cells by generating reactive oxygen species (ROS). Figure 27 The results showed that PtHP NP could induce the highest level of ROS in tumor tissue, which is consistent with its best therapeutic effect. High expression of ROS is usually associated with high cell apoptosis. Figure 28 The results of cell apoptosis showed that PtHP NP could induce the strongest cell apoptosis, which was consistent with its therapeutic effect and the level of induced ROS production. The effect of the drug on CSCs in tumor tissue was also tested. Figure 29 The results showed that PtHP NPs could reduce the expression of CD44 in tumor tissues. + CD24 - In addition, the effect of PtHP NP on tumor immunity was also tested. Figure 30 It can be seen that PtHP NP can increase the tumor infiltrating CD3 + T lymphocytes and can increase CD3 + CD4 in T lymphocytes+ CD8 + T cell ratio, which helps the body's immune response.

[0143] In summary, the results of the in vivo anti-tumor evaluation of PtHP NPs showed that PtHP NPs can induce high levels of ROS in tumors, leading to a high proportion of apoptosis, have a good tumor inhibition effect, and have good biological safety.

[0144] Those skilled in the art will easily understand that the above description is only a preferred embodiment of the present application, and is not intended to limit the present application, and any modifications, equivalent replacements and improvements made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A nano-drug delivery system based on an amphiphilic macromolecular compound, characterized in that, The amphiphilic macromolecular compound comprises a hydroxyalkyl starch and a photosensitizer, and the photosensitizer is a hydrophobic porphyrin heterocyclic macrocyclic organic compound. The amphiphilic macromolecular compound is obtained by coupling a photosensitizer and a hydroxyalkyl starch through a chemical bond, and the photosensitizer is a hydrophobic porphyrin heterocyclic macrocyclic organic compound. The hydroxyalkyl starch-platinum (IV) complex is prepared by the following method: (1) reacting cisplatin with hydrogen peroxide at 45-60°C to generate platinum (IV)-(OH)2; (2) reacting the platinum (IV)-(OH)2 obtained in step (1) with succinic anhydride to generate an axial carboxylic acid coordinated platinum (IV) complex platinum (IV)-(COOH)x, 1.0≤x<2.0; (3) reacting the axial carboxylic acid coordinated platinum (IV) complex platinum (IV)-(COOH)x, 1.0≤x<2.0, obtained in step (2) with a hydroxyalkyl starch in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine to generate a hydroxyalkyl starch-platinum (IV) complex.

2. The nano drug delivery system according to claim 1, wherein, The hydroxyalkyl starch is hydroxymethyl starch, hydroxyethyl starch, hydroxypropyl starch, or hydroxybutyl starch.

3. The nano drug delivery system according to claim 1, wherein, The chemical bond is an amide bond or an ester bond.

4. The nano drug delivery system according to claim 1, wherein, The preparation method of the amphiphilic macromolecular compound comprises the following steps: reacting a photosensitizer and a hydroxyalkyl starch in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine to generate a hydroxyalkyl starch-photosensitizer conjugate. The molecular weight of the hydroxyalkyl starch is 25-480 kDa, and the molar substitution degree of the hydroxyalkyl group is 0.4-0.

6.

5. The NDS of claim 4, wherein, The hydroxyalkyl starch-photosensitizer conjugate and the hydroxyalkyl starch-platinum (IV) complex are mixed and then the nano-drug delivery system is prepared by emulsification, dialysis, or high-pressure homogenization.

6. The NDS of claim 5, wherein, The mass ratio of the photosensitizer in the hydroxyalkyl starch-photosensitizer conjugate to the platinum element in the hydroxyalkyl starch-platinum (IV) complex is 1: (1-5).

7. The nano-drug delivery system of any one of claims 1-6 for use in the preparation of a medicament for treating and / or preventing cancer.

8. An anticancer drug, characterized by, The nano-drug delivery system of any one of claims 1-6 and a pharmaceutically acceptable additive.

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