Improved metal-polyphenol network-based dual-metabolism regulation type tumor-targeted nano drug-loaded preparation as well as preparation method and application of dual-metabolism regulation type tumor-targeted nano drug-loaded preparation

Abstract

The invention belongs to the technical field of pharmaceutical preparations and biological medicines, and relates to a double-metabolism regulation type tumor targeting nano drug-loaded preparation based on an improved metal-polyphenol network as well as a preparation method and application of the double-metabolism regulation type tumor targeting nano drug-loaded preparation. The medicine is formed by coating a hydrophobic medicine with an improved metal-polyphenol network carrier, and the hydrophobic medicine comprises a DHODH inhibitor and a DGAT1 inhibitor. The improved metal-polyphenol network carrier is formed by cross-linking and self-assembling iron ions and a polyphenol composite material, and the polyphenol composite material is formed by connecting hyaluronic acid and tannic acid through a linking group containing a disulfide bond. According to the tumor-targeted nano drug-loaded preparation provided by the invention, cell metabolism is regulated by entrapping the DHODH inhibitor and the DGAT1 inhibitor, a ferroptosis defense system is synchronously destroyed, and the lipid droplet level is reduced, so that cell apoptosis drug resistance generally existing in tumor chemotherapy is avoided.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical preparations and biomedicine, and relates to a dual-metabolic-regulation tumor-targeting nano-drug delivery system based on an improved metal-polyphenol network, and a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art that is already known in any country in the world.

[0003] Chemotherapy is one of the cornerstones of cancer treatment, but its efficacy is often severely compromised by drug resistance of tumor cells. Among them, the resistance to apoptosis is one of the most common and intractable problems. Tumor cells can escape from apoptotic death through various mechanisms (such as overexpression of anti-apoptotic proteins (such as Bcl-2 family), mutation or inactivation of key molecules in the apoptosis signaling pathway, enhancement of drug efflux pumps, etc.), leading to failure of chemotherapeutic drugs, increased risk of tumor recurrence and metastasis. Therefore, it is of urgent and great clinical significance to develop new tumor treatment strategies that can overcome the resistance to apoptosis. SUMMARY

[0004] In order to solve the problems of the existing tumor chemotherapy technology, the purpose of the present application is to provide a dual-metabolic-regulation tumor-targeting nano-drug delivery system based on an improved metal-polyphenol network, and a preparation method and application thereof. The tumor-targeting nano-drug delivery system provided by the present application encapsulates dihydroorotate dehydrogenase (DHODH) inhibitors and DGAT1 (diacylglycerol acyltransferase-1) inhibitors to regulate cell metabolism, simultaneously destroy the ferroptosis defense system and down-regulate the lipid droplet level, thereby avoiding the common cell apoptosis resistance in tumor chemotherapy.

[0005] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows: In a first aspect, an improved metal-polyphenol network carrier is formed by self-assembly of iron ions and polyphenol composite materials through mutual cross-linking, wherein the polyphenol composite material is formed by connecting hyaluronic acid and tannic acid through a connecting group containing a disulfide bond.

[0006] The metal-polyphenol network carrier of the present application connects hyaluronic acid and tannic acid through a linking group containing a disulfide bond to form an improved polyphenol. Under the action of the disulfide bond, the carrier can be destroyed in the high glutathione environment of the tumor and consume glutathione in the tumor microenvironment. In addition, the phenolic hydroxyl groups of tannic acid can chelate with iron ions to form a metal polyphenol network, and the tumor microenvironment in which tumor cells are located is generally acidic conditions, and the chelation between iron ions and phenolic hydroxyl groups of the metal polyphenol network formed by the present application will be destroyed under acidic conditions, releasing iron ions and the loaded drug. The released iron ions can mediate a Fenton-like reaction to produce a large amount of active oxygen, triggering cellular ferroptosis.

[0007] In another aspect, a preparation method of the improved metal-polyphenol network carrier described above, comprising the following steps: amidating hyaluronic acid with cystamine to obtain modified hyaluronic acid containing free primary amino groups and connected disulfide bonds; condensing tannic acid with the free amino groups of the modified hyaluronic acid to obtain a polyphenol composite material; mixing an iron salt with the polyphenol composite material to allow the iron ions and the polyphenol composite material to self-assemble into a metal-polyphenol network through mutual crosslinking, and obtaining the same.

[0008] The present application first reacts cystamine with hyaluronic acid to improve the grafting rate of disulfide bonds on the hyaluronic acid molecule.

[0009] In a third aspect, a dual metabolic regulation type tumor-targeting nano-drug delivery preparation based on an improved metal-polyphenol network is composed of the improved metal-polyphenol network carrier described above and a hydrophobic drug, and the hydrophobic drug includes a DHODH inhibitor and a DGAT1 inhibitor.

[0010] The DHODH inhibitor is related to processes such as cellular ferroptosis, pyrimidine biosynthesis, and mitochondrial respiratory chain, and can affect tumor cell metabolism in multiple ways. The action of the DGAT1 inhibitor includes promoting the biosynthesis of triglycerides to provide essential energy supply for the abnormal proliferation of tumor cells; at the same time, the enzyme mediates the formation of lipid droplets, thereby effectively storing excess lipids, protecting excess polyunsaturated fatty acids in cells from active oxygen damage and lipid peroxidation, and isolating hydrophobic drugs in cells, which is beneficial to tumor survival.

[0011] The present application combines the improved metal-polyphenol network carrier with the DHODH inhibitor and the DGAT1 inhibitor, and after administration to the subject, the hyaluronic acid on the surface of the tumor-targeting nano-drug delivery preparation can not only endow the tumor-targeting nano-drug delivery preparation with tumor-specific targeting ability, but also avoid unnecessary adhesion of tannic acid to other proteins outside the target during delivery. The addition of disulfide bonds can accelerate the degradation of the nano-drug delivery preparation while consuming glutathione in the tumor microenvironment. As an important component of the carrier, the dissociated iron ions under the acidic conditions of the tumor can continuously generate hydroxyl radicals through the cycle of valence states, consume glutathione peroxidase 4 (GPX4) in tumor cells, induce the peroxidation of polyunsaturated fatty acids in tumor cells, and initiate ferroptosis. The DHODH inhibitor can destroy the DHODH-mediated ferroptosis defense mechanism in cells and induce more severe ferroptosis under the condition of GSH / glutathione peroxidase 4 (GPX4) inhibition caused by iron ions. At the same time, due to the key role of DHODH in the pyrimidine synthesis process of the tumor, the DHODH inhibitor can also cause cell cycle arrest and delay tumor progression by inducing depletion of the pyrimidine pool. In addition, the DGAT1 inhibitor in the tumor-targeting nano-drug delivery preparation can inhibit the increase of lipid droplets caused by the induction of cell cycle arrest by the DHODH inhibitor and the subsequent resistance to ferroptosis, and the down-regulation of lipid droplet level is also conducive to hindering the proliferation, migration and subsequent development of the tumor.

[0012] In a fourth aspect, a preparation method of the above-mentioned tumor-targeting nano-drug delivery preparation based on the improved metal-polyphenol network for dual metabolic regulation, comprises the preparation method of the above-mentioned improved metal-polyphenol network carrier. The DHODH inhibitor, the DGAT1 inhibitor, the iron salt and the polyphenol composite material are mixed and reacted, so that the iron ions and the polyphenol composite material are self-assembled into a metal-polyphenol network through mutual crosslinking, and the DHODH inhibitor and the DGAT1 inhibitor are coated, and the preparation is obtained.

[0013] In a fifth aspect, the above-mentioned tumor-targeting nano-drug delivery preparation based on the improved metal-polyphenol network for dual metabolic regulation is applied to the preparation of a drug for treating tumors.

[0014] The present application has the following advantages: (1) The present application uses the chelation between disulfide bonds, phenolic hydroxyl groups and iron ions to construct the carrier of the nano-drug delivery preparation, which has the effect of specifically degrading the drug in the tumor microenvironment.

[0015] (2) The hyaluronic acid on the surface of the carrier constructed by the present application can realize tumor-specific active targeting.

[0016] (3) The nano drug delivery preparation provided by the application realizes controllable co-delivery of iron death inducers and metabolic regulators. The nano drug delivery preparation can not only directly induce iron death through Fenton reaction catalysis of lipid peroxidation, but also comprehensively activate cell iron death by synergistically regulating lipid metabolism and DHODH axis related metabolic pathways, thereby providing a new strategy for tumor treatment.

[0017] (4) The application has simple preparation process, strong practicability and easy popularization. BRIEF DESCRIPTION OF DRAWINGS

[0018] The drawings accompanying the specification of the application form a part of the application and serve to further understand the application. The illustrative embodiments of the application and their description serve to explain the application without constituting an improper limitation thereof.

[0019] Figure 1 A flowchart for preparing AB@HA-TA / Fe for the embodiments of the application; Figure 2 A preparation route diagram for preparing HA-TA for the embodiments of the application; Figure 3 H NMR spectra of HA-SS and HA prepared in Example 1 of the application; 1 H NMR spectra of TA, HA-SS and HA-TA prepared in Example 1 of the application; Figure 4 H NMR spectra of TA, HA-SS and HA-TA prepared in Example 1 of the application; 1 H NMR spectra of TA, HA-SS and HA-TA prepared in Example 1 of the application; Figure 5 High performance liquid chromatograms of A922500 (A), BQR (B), HA-TA / Fe (C) and AB@HA-TA / Fe (D) in Example 1 of the application; Figure 6 TEM images of AB@HA-TA / Fe prepared in Example 1 of the application under the conditions of pH=7.4 (A) or pH=6.0, 10 mM GSH (B).

[0020] Figure 7 Cumulative release curves of BQR (A) and A922500 (B) of AB@HA-TA / Fe prepared in Example 1 of the application under the conditions of pH=7.4 or pH=6.0, 10 mM GSH.

[0021] Figure 8 Confocal images and flow cytometry results of 4T1 cells after different time treatments on free Ce6 and Ce6@HA-TA / Fe, A is the confocal image, B is the flow cytometry result image, C is the mean fluorescence intensity (MFI) quantification image in the flow cytometry result; Figure 9The real-time fluorescence images in vivo after injecting different preparations into the mice of the present application via the caudal vein and the fluorescence images of the organs and tumors ex vivo after 24 h, (A) the in vivo imaging results of the tumor-bearing mice at different time points after administration of free Ce6 and Ce6@HA-TA / Fe, the ex vivo imaging results (B) and the radiation efficiency (C) of the tumors and main organs of the mice after 24 h of administration. DETAILED DESCRIPTION

[0022] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0023] It is also important to note that the terms used herein are not intended to limit the particular embodiments of the present application which can be practiced with the examples of the present application. As used herein, unless otherwise clear from context, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, components, and / or groups thereof, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0024] In view of the influence of the apoptosis resistance commonly existing in the existing tumor chemotherapy on the effect of the tumor chemotherapy, the present application provides a double-metabolic regulation type tumor targeting nano drug preparation based on a modified metal-polyphenol network and a preparation method and application thereof.

[0025] In a typical embodiment of the present application, a modified metal-polyphenol network carrier is provided, which is formed by self-assembly of iron ions and a polyphenol composite material through mutual crosslinking, and the polyphenol composite material is formed by connecting hyaluronic acid and tannic acid through a connecting group containing a disulfide bond.

[0026] In some embodiments, in the polyphenol composite material, the degree of substitution of tannic acid in hyaluronic acid is 1.15-6.25.

[0027] In some embodiments, the weight average molecular weight of the hyaluronic acid is 1000-10000 Da.

[0028] In another embodiment of the present application, a preparation method of the above-mentioned modified metal-polyphenol network carrier is provided, which comprises the following steps: The hyaluronic acid is subjected to an amidation reaction with cystamine to obtain modified hyaluronic acid containing free primary amino groups and connected disulfide bonds; The tannic acid is subjected to a condensation reaction with the free amino groups of the modified hyaluronic acid to obtain a polyphenol composite material; The iron salt is mixed with the polyphenol composite material, so that the iron ions and the polyphenol composite material are self-assembled to form a metal-polyphenol network through mutual crosslinking, and the preparation is obtained.

[0029] The ferric salt refers to a compound with a trivalent iron cation, such as ferric chloride, ferric sulfate, ferric nitrate, etc.

[0030] In some embodiments, EDCI (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) are used for catalytic activation during the amidation reaction. Specifically, the temperature of the amidation reaction is room temperature, and the reaction time is 22-26 h. The room temperature in the present application refers to the temperature of the indoor environment, which is generally 15-30 ℃.

[0031] In some embodiments, the mass ratio of hyaluronic acid to cystamine is 1:1.8-1.9.

[0032] In some embodiments, the mass ratio of modified hyaluronic acid to tannic acid is 1:1.5-10.0, preferably 1:8.0-10.0, and further preferably 1:8.0-9.0.

[0033] In some embodiments, the pH of the condensation reaction is 8.0-9.0.

[0034] In some embodiments, the temperature of the condensation reaction is room temperature, and the time is 5-7 h.

[0035] In some embodiments, the mixing method of the ferric salt and the polyphenol composite material is vortex.

[0036] The third embodiment of the present application provides a double-metabolic regulation type tumor targeting nano-drug preparation based on an improved metal-polyphenol network, which is composed of a hydrophobic drug coated by the improved metal-polyphenol network carrier, and the hydrophobic drug includes a DHODH inhibitor and a DGAT1 inhibitor.

[0037] In some embodiments, the DHODH inhibitor is Brequinar. The full name of Brequinar is "Brequinar", and the abbreviation is BQR. The drug loading and encapsulation efficiency of BQR are 1.80-6.00% and 8.50-19.50%, respectively, and preferably 3.55±0.7% and 12.78±1.42%.

[0038] In some embodiments, the DGAT1 inhibitor is A922500. The CAS number of A922500 is 959122-11-3. The drug loading and encapsulation efficiency of A922500 are 0.30-16.00% and 3.00-18.50%, respectively, and preferably 5.98±1.3% and 10.76±1.06%.

[0039] In some embodiments, the mass ratio of HA-TA to DHODH inhibitor, DGAT1 inhibitor in the improved metal-polyphenol network carrier is 39-41:1:0.25-4, preferably 39-41:1:1.8-2.2.

[0040] In some embodiments, the particle size of the nano-drug delivery preparation is 185.0 nm-356.0 nm, preferably 305.7±22.60 nm.

[0041] In some embodiments, the surface potential of the nano-drug delivery preparation is -21.9 mV to -28.0 mV, preferably -25.2±0.737 mV.

[0042] The fourth embodiment of the present application provides a preparation method of the above-mentioned double-metabolic-regulation type tumor-targeting nano-drug delivery preparation based on the improved metal-polyphenol network, comprising the preparation method of the improved metal-polyphenol network carrier mentioned above. The DHODH inhibitor, the DGAT1 inhibitor, and the iron salt and the polyphenol composite material are mixed and reacted, so that the iron ions and the polyphenol composite material are self-assembled to form a metal-polyphenol network by mutual crosslinking, and the DHODH inhibitor and the DGAT1 inhibitor are coated, and the improved metal-polyphenol network carrier is obtained.

[0043] The fifth aspect is the use of the above-mentioned double-metabolic-regulation type tumor-targeting nano-drug delivery preparation based on the improved metal-polyphenol network in the preparation of a drug for treating tumors.

[0044] In some embodiments, the tumor is breast cancer.

[0045] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific examples and comparative examples.

[0046] Example 1 A preparation method of a double-metabolic-regulation type tumor-targeting nano-drug delivery preparation based on an improved metal-polyphenol network (named AB@HA-TA / Fe), comprising the following steps: 1. Preparation of modified hyaluronic acid (HA-SS) with disulfide bond 500 mg of ultra-low molecular weight hyaluronic acid (HA, about 9 kDa) was dissolved in 50 mL of 10 mM phosphate buffer (PBS), and then 1.875 mmol of EDCI and 1.875 mmol of NHS were added in sequence after stirring and hydrating. After stirring for 1 h, 3.98 mmol of cystamine dihydrochloride was added, and after stirring at room temperature for 24 h, it was placed in a 3.5 kDa dialysis bag for three days of water dialysis. After freeze-drying, HA-SS was obtained, as shown in Figure 3 The preparation process is as shown in Figure 2as shown.

[0047] 2. Preparation of polyphenol composite (HA-TA) Dissolve 425 mg tannic acid (TA) and 50 mg HA-SS in 6 mL PBS and 4 mL PBS respectively, mix under stirring, adjust the pH of the solution to 8.5, stir the reaction at room temperature for 6 h, then place the reaction solution in a 3.5 kDa dialysis bag, dialyze in 25% (v / v) ethanol for 1 day and in distilled water for 2 days to remove unreacted raw materials, and obtain HA-TA after freeze-drying, as shown in Figure 4 , and the preparation process is as shown in Figure 1 , 2 .

[0048] 3. Preparation of AB@HA-TA / Fe Weigh an appropriate amount of FeCl3·6H2O and prepare a 10 mg / mL solution with deionized water for standby. Dissolve an appropriate amount of BQR and A922500 in DMSO to prepare a concentrated solution, take 0.5 mg BQR and 1.0 mg A922500 containing DMSO concentrated solution into 6.5 mL solution containing 20 mg HA-TA, vortex to mix, then add 120 μL of 10 mg / mL FeCl3·6H2O solution, vortex for 50 min, then centrifuge (12000 rpm, 15 min) to obtain the precipitate, and then wash the precipitate with DMSO and water mixture and deionized water respectively until it is colorless, and dry the obtained precipitate to obtain AB@HA-TA / Fe, as shown in Figure 5 , and the preparation process is as shown in Figure 1 . After the prepared AB@HA-TA / Fe is incubated in two media of pH=7.4 and pH=6.0+10 mM GSH for 24 h, it is added dropwise to a copper mesh, dried, and then the microstructure of the nanoparticles is observed by TEM, and the results are as shown in Figure 6 .

[0049] Example 2 This example is the same as Example 1, except that: 2. Preparation of polyphenol composite (HA-TA) Dissolve 425 mg tannic acid (TA) and 50 mg HA-SS in 6 mL PBS and 4 mL PBS respectively, mix under stirring, adjust the pH of the solution to 8.5, stir the reaction at room temperature for 6 h, then place the reaction solution in a 3.5 kDa dialysis bag, dialyze in 25% (v / v) ethanol for 1 day and in distilled water for 2 days to remove unreacted raw materials, and obtain HA-TA after freeze-drying, as shown in

[0050] Example 3 This example is the same as example 1, except that: 2. Preparation of polyphenol composite (HA-TA) 212.5 mg of tannic acid (TA) and 50 mg of HA-SS were dissolved in 6 mL of PBS and 4 mL of PBS, respectively, mixed under stirring, the pH of the solution was adjusted to 8.5, and the reaction was stirred at room temperature for 6 h. Then the reaction solution was placed in a 3.5 kDa dialysis bag and dialyzed in 25% (v / v) ethanol for 1 day and in distilled water for 2 days to remove unreacted raw materials. After lyophilization, HA-TA was obtained.

[0051] Example 4 This example is the same as example 1, except that: An appropriate amount of FeCl3·6H2O was weighed and prepared into a 10 mg / mL solution with deionized water for standby. An appropriate amount of BQR and A922500 was dissolved in DMSO to prepare a concentrated solution. 0.5 mg of BQR and 2.0 mg of A922500 contained in the DMSO concentrated solution were added to 6.5 mL of a solution in which 20 mg of HA-TA was dissolved, and vortexed to mix. Then 120 μL of 10 mg / mL FeCl3·6H2O solution was added, and vortexed for 50 min. The precipitate was obtained by centrifugation (12000 rpm, 15 min), and the precipitate was washed with a mixture of DMSO and water and deionized water in sequence until it was colorless. The obtained precipitate was dried to obtain AB@HA-TA / Fe.

[0052] Example 5 This example is the same as example 1, except that: An appropriate amount of FeCl3·6H2O was weighed and prepared into a 10 mg / mL solution with deionized water for standby. An appropriate amount of BQR and A922500 was dissolved in DMSO to prepare a concentrated solution. 0.5 mg of BQR and 0.5 mg of A922500 contained in the DMSO concentrated solution were added to 6.5 mL of a solution in which 20 mg of HA-TA was dissolved, and vortexed to mix. Then 120 μL of 10 mg / mL FeCl3·6H2O solution was added, and vortexed for 50 min. The precipitate was obtained by centrifugation (12000 rpm, 15 min), and the precipitate was washed with a mixture of DMSO and water and deionized water in sequence until it was colorless. The obtained precipitate was dried to obtain AB@HA-TA / Fe.

[0053] Example 6 This example is the same as example 1, except that: A certain amount of FeCl3·6H2O was weighed and prepared into a 10 mg / mL solution with deionized water for standby. A certain amount of BQR and A922500 was dissolved in DMSO to prepare a concentrated solution. The DMSO concentrated solution containing 0.5 mg of BQR and 0.25 mg of A922500 was added to 6.5 mL of the solution in which 20 mg of HA-TA was dissolved, and vortexed to mix. Then, 120 μL of the 10 mg / mL FeCl3·6H2O solution was added, and vortexed for 50 min. After centrifugation (12000 rpm, 15 min), the precipitate was obtained, and was washed with a DMSO and water mixture and deionized water in sequence until the precipitate was colorless. The obtained precipitate was dried to obtain AB@HA-TA / Fe.

[0054] Example 7 This example is the same as Example 1, except that: A certain amount of FeCl3·6H2O was weighed and prepared into a 10 mg / mL solution with deionized water for standby. A certain amount of BQR and A922500 was dissolved in DMSO to prepare a concentrated solution. The DMSO concentrated solution containing 0.5 mg of BQR and 0.25 mg of A922500 was added to 6.5 mL of the solution in which 20 mg of HA-TA was dissolved, and vortexed to mix. Then, 120 μL of the 10 mg / mL FeCl3·6H2O solution was added, and vortexed for 50 min. After centrifugation (12000 rpm, 15 min), the precipitate was obtained, and was washed with a DMSO and water mixture and deionized water in sequence until the precipitate was colorless. The obtained precipitate was dried to obtain AB@HA-TA / Fe.

[0055] The performance test was as follows: 1. Evaluation of in vitro release behavior of AB@HA-TA / Fe: After AB@HA-TA / Fe was dispersed, 1 mL was taken and loaded into a dialysis bag with a molecular weight cut-off of 3.5 kDa. The two ends were tied with cotton thread. Three groups of samples were prepared in parallel for each release medium to determine the release condition. The dialysis bag was immersed in 9 mL of release medium, and the whole system was placed in a constant temperature water bath oscillator (37°C, 100 rpm) for continuous oscillation for 48 h to simulate the drug release behavior under physiological conditions. At the set time point, 1 mL of release medium was taken out and supplemented with the same volume of fresh release medium to maintain the leak condition. The sample was filtered through a 0.22 μm microporous filter membrane, and the content of BQR and A922500 in the release medium at each time point was detected by HPLC. The cumulative release amount Q was calculated according to the following formula, and the cumulative release curve was drawn, as shown in Figure 7

[0056]

[0057] ​Cellular uptake experiment: Because BQR and A922500 are inherently nonfluorescent, chlorine e6 (Ce6) was used as a fluorescent tracer to investigate cellular nanoparticle uptake. Ce6-loaded Ce6@HA-TA / Fe was prepared following the same preparation steps as AB@HA-TA / Fe. 4T1 cells were evenly seeded onto slides in sterile 12-well plates. After overnight incubation, cells were incubated with free Ce6 and Ce6@HA-TA / Fe (Ce6 concentration was 5 μg / mL) for various times (1, 2, and 4 h). After incubation, the cells were washed with PBS, fixed with 4% paraformaldehyde, stained with DAPI, and mounted with anti-fading mounting media. Ce6 fluorescence in the cells was observed under a confocal laser scanning microscope (CLSM). To quantitatively observe the uptake of nanoparticles by 4T1 cells, 4T1 cells were cultured in 12-well plates and incubated with drugs according to the above protocol. After incubation, the drugs were washed away with PBS, and the cells were digested and collected, and the mean fluorescence intensity (MFI) in the cells was quantified using flow cytometry (FCM). The results are shown in Figure 2. Figure 8 shown.

[0058] Nanoparticle in vivo distribution assay: To establish a tumor-bearing mouse model, female Balb / c mice (6-8 weeks old) were subcutaneously injected with 1×106 cells in the fourth mammary fat pad on the right side. One week after tumor cell inoculation, the mice were allowed to grow to a tumor volume of 100-120 mm. 3 Mice were randomly divided into two groups, three in each group, and injected with free Ce6 or Ce6@HA-TA / Fe (Ce6 content 5 mg / kg) through the tail vein. In vivo imaging was performed on the mice at 1, 2, 4, 8, 12, and 24 hours. After 24 hours, the mice were sacrificed, and their tumors and major organs (including heart, liver, spleen, lungs, and kidneys) were completely isolated. Immediately afterwards, ex vivo imaging of each tissue sample was performed. The results are shown in Figure 2. Figure 9 shown.

[0059] Result Analysis HA-SS prepared in Example 1 1 H NMR Figure 3 As shown in Figure 3, the two methylene proton peaks of the cystamine structure in HA-SS appeared at chemical shifts of approximately 3.4 ppm and 3.0 ppm, indicating that cystamine was successfully grafted onto HA.

[0060] HA-TA prepared in Example 1 1 H NMR Figure 4As shown, the results show that the synthesized HA-TA has a characteristic peak at a position of about 7.0 ppm (as indicated by an arrow), in addition to a plurality of characteristic peaks corresponding to HA-SS, which is just the characteristic peak of TA at this position, proving that TA is successfully grafted with HA-SS to form HA-TA. The TA substitution degree in HA-TA is as shown in Table 1. The substitution degree (DS) is calculated according to the following formula:

[0061] In the formula, m0 is the mass of TA in HA-TA detected by ultraviolet absorption, m is the mass of HA-TA, M TA is the relative molecular weight of TA, and M HA is the relative molecular weight of HA.

[0062] Table 1 TA substitution degree in HA-TA prepared in Examples 1-3

[0063] Table 1 shows that the TA substitution degree in the HA-TA prepared in Example 1 is higher, and therefore, the HA-TA prepared in Example 1 is used for subsequent experiments.

[0064] The particle size, surface potential, drug loading and encapsulation efficiency of AB@HA-TA / Fe prepared in different examples are shown in Table 2.

[0065] Table 2 Particle size, surface potential, drug loading and encapsulation efficiency of AB@HA-TA / Fe prepared in different examples

[0066] Table 2 shows that the AB@HA-TA / Fe prepared in Example 1 has a higher drug loading and encapsulation efficiency for BQR and A922500.

[0067] The micro-morphology of AB@HA-TA / Fe after incubation in pH=7.4 and pH=6.0, 10 mM GSH release medium for 24 h was investigated. AB@HA-TA / Fe can maintain good stability and dispersity under the condition of pH=7.4, and AB@HA-TA / Fe presents a spherical shape with uniform particle size, as shown in Figure 6 A. However, after incubation in pH=6.0, 10 mM GSH for 24 h, the complete structure of the nanoparticles is destroyed and serious adhesion occurs, proving that AB@HA-TA / Fe will degrade under acidic and GSH conditions, as shown in Figure 6 B.

[0068] The in vitro drug release behavior of the nanoparticles was detected by dialysis bag method. AB@HA-TA / Fe released the drug quickly under the condition of pH=6.0+10 mM GSH, and about 40% of the two drugs were released after 48 h. In contrast, AB@HA-TA / Fe released the drug slowly under the condition of pH=7.4, and only about 25% of the drug was released after 48 h. This may be due to the destruction of the complexation between TA and Fe in HA-TA / Fe and the rapid destruction of the disulfide bond in HA-TA under high GSH conditions, which leads to the accelerated release of the loaded drug, as shown in Figure 7 .

[0069] Confocal and flow cytometry were used to detect the difference in the uptake ability of cells to free drugs and nanoparticles. From the CLSM result graph, Ce6@HA-TA / Fe had the most obvious red fluorescence in tumor cells after 4 h of incubation, which proved that Ce6@HA-TA / Fe could be taken up into 4T1 cells. Further FCM results Figure 8 A and quantitative results Figure 8 B show that with the extension of incubation time, the fluorescence intensity in both free Ce6 group and Ce6@HA-TA / Fe treated group cells is continuously enhanced, which proves the time dependence of cell drug uptake. In addition, from the 1st hour, the cells have a significantly better uptake ability for Ce6@HA-TA / Fe than for free Ce6; and after the same incubation time (1, 2, 4 h), the MFI in the Ce6@HA-TA / Fe treated group is significantly higher than that in the free Ce6 group, indicating that HA-TA / Fe can effectively enhance the uptake of nanoparticles by tumor cells. The reason why the nanoparticle preparation has better uptake than the free drug may be due to the endocytosis mediated by the interaction between the HA on the surface of the nanoparticles and the CD44 receptors on the tumor surface.

[0070] The present application uses Ce6 as a model drug and a fluorescent tracer to study the accumulation of free drugs and nanoparticles in vivo. The in vivo imaging results of mice Figure 9 A show that the free drug has poor targeting in vivo distribution, and it is quickly cleared in about 12 h. In contrast to the free drug, Ce6@HA-TA / Fe can better accumulate in the tumor site, and reaches the maximum fluorescence intensity in about 8 h, and the fluorescence of the nanoparticles accumulated in the tumor site can still be observed after 24 h. The mice were sacrificed after 24 h to separate the tumor and main organs for fluorescence imaging, and the results are shown in Figure 9 B and Figure 9As shown in Fig. 1C, it can be seen that free Ce6 has less accumulation in tumor site and more accumulation in liver site, suggesting that liver may be the main way of Ce6 metabolism. The tumor of mice in Ce6@HA-TA / Fe treatment group shows stronger fluorescence, and its fluorescence intensity is 2.35 times that of the free Ce6 treatment group, with significant difference. The above results prove that the nanoparticles can effectively enhance the accumulation of therapeutic drugs in the tumor site, which may be related to the tumor targeting effect mediated by HA on the surface of the nanoparticles, and is conducive to better tumor targeted therapy.

[0071] The above merely describes preferred embodiments of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An improved metal-polyphenol network carrier, characterized in that: The composite material is formed by cross-linking and self-assembly of iron ions and polyphenol composite materials, wherein the polyphenol composite material is formed by connecting hyaluronic acid and tannic acid through a connecting group containing a disulfide bond.

2. The improved metal-polyphenol network carrier according to claim 1, characterized in that: In the polyphenol composite material, the degree of substitution of tannic acid in hyaluronic acid is 1.15-6.

25.

3. The improved metal-polyphenol network carrier according to claim 1, characterized in that The weight average molecular weight of the hyaluronic acid is 1000-10000 Da.

4. A method for preparing the improved metal-polyphenol network carrier according to claim 1, characterized in that: The steps include: The modified hyaluronic acid containing free primary amino groups and connected with disulfide bonds is obtained by subjecting hyaluronic acid to an amidation reaction with cystamine; carrying out a condensation reaction between tannic acid and the free amino groups of the modified hyaluronic acid to obtain a polyphenol composite material; The iron salt is mixed with the polyphenol composite material, so that the iron ions and the polyphenol composite material are self-assembled through mutual cross-linking to form a metal-polyphenol network.

5. The method for preparing the improved metal-polyphenol network carrier according to claim 4, wherein During the amidation reaction, EDCI and NHS were used for catalytic activation; Alternatively, the mass ratio of hyaluronic acid to cystamine is 1:1.8-1.9; Alternatively, the mass ratio of modified hyaluronic acid to tannic acid is 1:1.5-10.0, preferably 1:8.0-10.0; Alternatively, the pH of the condensation reaction is 8.0 to 9.0; Alternatively, the condensation reaction temperature is room temperature and the time is 5 to 7 hours.

6. A dual metabolism-regulated tumor-targeted nano-drug delivery formulation based on an improved metal-polyphenol network, characterized by: The improved metal-polyphenol network carrier according to any one of claims 1 to 3 is coated with a hydrophobic drug, wherein the hydrophobic drug comprises a DHODH inhibitor and a DGAT1 inhibitor.

7. The dual metabolism-regulated tumor-targeted nano-drug delivery formulation based on the improved metal-polyphenol network according to claim 6, characterized in that: The DHODH inhibitor is brequinar; or, the DGAT1 inhibitor is A922500; Alternatively, the mass ratio of HA-TA to DHODH inhibitor and DGAT1 inhibitor in the improved metal-polyphenol network carrier is 39-41:1:1.8-2.

2.

8. A method for preparing the dual metabolism-regulated tumor-targeted nano-drug-carrying preparation based on the improved metal-polyphenol network according to claim 6 or 7, characterized in that: The preparation method of the improved metal-polyphenol network carrier according to claim 4 or 5; The DHODH inhibitor and the DGAT1 inhibitor are mixed with the iron salt and the polyphenol composite material to react, so that the iron ions and the polyphenol composite material are self-assembled through mutual cross-linking to form a metal-polyphenol network, and the DHODH inhibitor and the DGAT1 inhibitor are coated to obtain the obtained product.

9. Use of the improved metal-polyphenol network-based dual metabolism-regulated tumor-targeted nano-drug-carrying preparation according to claim 6 or 7 in the preparation of drugs for treating tumors.

10. The use according to claim 9, characterized in that: The tumor is breast cancer.

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