Metal polyphenol network-encapsulated calcium peroxide nanoparticles, and preparation method and application thereof

Abstract

The application provides a metal polyphenol network-wrapped calcium peroxide nanoparticle and a preparation method and application thereof, and belongs to the technical field of tumor treatment. The metal polyphenol network-wrapped calcium peroxide nanoparticle has a protective effect on unstable calcium peroxide nanoparticles; the released metal ions catalyze hydrogen peroxide to generate hydroxyl radicals under acidic conditions, and then kill tumor cells, realize chemical kinetic therapy, can also consume glutathione, enhance the accumulation of active oxygen, and then enhance the anti-tumor effect; when dispersed into an acidic environment, the metal polyphenol network layer thereof can rapidly degrade, expose the calcium peroxide nanoparticles, and then explosively release a large amount of calcium ions and oxygen, which can more effectively activate calcium overload and relieve the tumor hypoxic microenvironment; by loading drugs, the effective accumulation of the drugs in the tumor site is improved, the combined treatment of chemical kinetic therapy and chemotherapy is realized, and thus the anti-tumor immune response is activated, and the anti-tumor effect is improved.

Description

Technical Field

[0001] This invention relates to the field of tumor treatment technology, and in particular to a metal polyphenol network-encapsulated calcium peroxide nanoparticle, its preparation method, and its application. Background Technology

[0002] Despite significant advancements in our understanding of tumorigenesis and cancer treatment, cancer still accounts for 30% of global deaths due to tumor heterogeneity and multidrug resistance. Surgery, radiotherapy, and chemotherapy have long been the three conventional treatments for cancer, but their efficacy remains unsatisfactory. Novel treatment strategies such as calcium overload therapy, chemokinetics, immunotherapy, photothermal therapy, and photodynamic therapy offer more avenues for cancer treatment. However, due to the complexity of tumors, relying solely on a single novel treatment strategy often has limitations and fails to achieve optimal therapeutic effects. Therefore, the combined application of treatment strategies with different mechanisms and targets has become an important means of improving efficacy.

[0003] Existing technologies, such as the patent with publication number CN113952361A, use a combination of Prussian blue and calcium peroxide to improve the therapeutic effect on tumors. However, its tumor treatment effect is expected to be further improved. Summary of the Invention

[0004] The purpose of this invention is to provide a metal polyphenol network-encapsulated calcium peroxide nanoparticle, its preparation method, and its application. The metal polyphenol network-encapsulated calcium peroxide nanoparticles prepared by this invention exhibit better tumor treatment effects.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for preparing calcium peroxide nanoparticles encapsulated in a metal polyphenol network, comprising the following steps:

[0007] (1) After mixing calcium peroxide nanoparticles, drugs and a first solvent, a chelation reaction was carried out to obtain drug-loaded calcium peroxide nanoparticles.

[0008] (2) After mixing the polymer solution and the polyphenol solution, a dehydration condensation reaction is carried out to obtain a polymer-modified polyphenol solution;

[0009] (3) The drug-loaded calcium peroxide nanoparticles obtained in step (1) are mixed with the second solvent, the polymer-modified polyphenol solution obtained in step (2) and the metal salt solution and then a coordination complexation reaction is carried out to obtain metal polyphenol network-encapsulated calcium peroxide nanoparticles.

[0010] The steps (1) and (2) are not in any particular order.

[0011] Preferably, in step (1), the mass ratio of calcium peroxide nanoparticles to drug is (10-55):1.

[0012] Preferably, the temperature of the chelation reaction in step (1) is 4 to 40°C, and the time of the chelation reaction is 1 to 48 hours.

[0013] Preferably, the mass ratio of the polymer in the polymer solution and the polyphenol in the polyphenol solution in step (2) is (1-3):1.

[0014] Preferably, the temperature of the dehydration condensation reaction in step (2) is 4 to 40°C, and the time of the dehydration condensation reaction is 1 to 48 hours.

[0015] Preferably, the molar ratio of polyphenols in the polyphenol solution of step (2) to metal ions in the metal salt solution of step (3) is (0.5-10):1.

[0016] Preferably, in step (3), the molar ratio of calcium peroxide in the drug-loaded calcium peroxide nanoparticles to the metal ions in the metal salt solution is (20-50):1.

[0017] Preferably, the temperature of the coordination complexation reaction in step (3) is 4 to 40°C, and the time of the coordination complexation reaction is 1 to 48 hours.

[0018] The present invention provides a metal polyphenol network-encapsulated calcium peroxide nanoparticle prepared by the preparation method described above, comprising drug-loaded calcium peroxide nanoparticles and a metal polyphenol network encapsulating the surface of the drug-loaded calcium peroxide nanoparticles.

[0019] The present invention also provides the application of the metal polyphenol network-encapsulated calcium peroxide nanoparticles described in the above technical solution in the preparation of tumor drugs.

[0020] This invention provides a method for preparing calcium peroxide nanoparticles encapsulated in a metal polyphenol network, comprising the following steps: (1) mixing calcium peroxide nanoparticles, a drug and a first solvent and performing a chelation reaction to obtain drug-loaded calcium peroxide nanoparticles; (2) mixing a polymer solution and a polyphenol solution and performing a dehydration condensation reaction to obtain a polymer-modified polyphenol solution; (3) mixing the drug-loaded calcium peroxide nanoparticles obtained in step (1) with a second solvent, the polymer-modified polyphenol solution obtained in step (2) and a metal salt solution and performing a coordination complexation reaction to obtain calcium peroxide nanoparticles encapsulated in a metal polyphenol network; steps (1) and (2) are not in any particular order. This invention involves mixing calcium peroxide nanoparticles with a drug to obtain drug-loaded calcium peroxide nanoparticles. These nanoparticles are then mixed with a metal salt solution and a polymer-modified polyphenol solution to undergo a coordination complexation reaction. This process forms an in-situ metal-polyphenol network on the surface of the drug-loaded calcium peroxide nanoparticles. The metal-polyphenols encapsulate the unstable calcium peroxide nanoparticles, preventing premature exposure and hydrolysis before reaching the lesion site and avoiding adverse reactions. Furthermore, the metal ions released from the metal-polyphenol-encapsulated calcium peroxide nanoparticles catalyze the generation of hydroxyl radicals from hydrogen peroxide under acidic conditions, thereby killing tumor cells and achieving chemokinetic therapy. Furthermore, metal ions can consume glutathione, enhance the accumulation of reactive oxygen species, and thus enhance the anti-tumor efficacy. When metal polyphenol-encapsulated calcium peroxide nanoparticles are dispersed in an acidic environment, their metal polyphenol network layer rapidly degrades, exposing the calcium peroxide nanoparticles and then explosively releasing a large amount of calcium ions and oxygen, which can more effectively activate calcium overload and alleviate the hypoxic microenvironment of the tumor. The metal polyphenol network-encapsulated drug-loaded calcium peroxide nanoparticles, by loading drugs, improve the effective accumulation of drugs at the tumor site, achieving combined chemotherapy and chemokinetics, thereby activating the anti-tumor immune response and improving the anti-tumor effect. The results of the examples show that the metal polyphenol network-encapsulated drug-loaded calcium peroxide nanoparticles prepared in this invention have excellent anti-breast cancer effects. Attached Figure Description

[0021] Figure 1 The particle size of the CaO2 nanoparticles prepared in step (1) of Example 1 and the drug-loaded calcium peroxide nanoparticles prepared in Examples 1 to 6;

[0022] Figure 2 The encapsulation efficiency and drug loading of the drug-loaded calcium peroxide nanoparticles prepared in Examples 1-6;

[0023] Figure 3 The particle size of the CaO2 nanoparticles prepared in step (1) of Example 7, and the drug-loaded calcium peroxide nanoparticles prepared in Examples 5 and 7-11;

[0024] Figure 4 The encapsulation efficiency and drug loading of the drug-loaded calcium peroxide nanoparticles prepared in Examples 5 and 7-11;

[0025] Figure 5 The particle size of the metal polyphenol network-encapsulated calcium peroxide nanoparticles prepared in Examples 12-18;

[0026] Figure 6 Macroscopic images of the calcium peroxide nanoparticle dispersion prepared in Example 12, the drug-loaded calcium peroxide nanoparticle dispersion prepared in Example 12, and the metal polyphenol network-encapsulated calcium peroxide nanoparticle dispersion prepared in Example 18, from left to right: the calcium peroxide nanoparticle dispersion prepared in Example 12, the drug-loaded calcium peroxide nanoparticle dispersion prepared in Example 12, and the metal polyphenol network-encapsulated calcium peroxide nanoparticle dispersion prepared in Example 18.

[0027] Figure 7 The particle size distributions are as follows: calcium peroxide nanoparticles prepared in step (1) of Example 12, drug-loaded calcium peroxide nanoparticles prepared in step (2) of Example 12, and metal polyphenol network-encapsulated calcium peroxide nanoparticles prepared in Example 18. In this distribution, A represents calcium peroxide nanoparticles prepared in step (1) of Example 12, B represents drug-loaded calcium peroxide nanoparticles prepared in step (2) of Example 12, and C represents metal polyphenol network-encapsulated calcium peroxide nanoparticles prepared in Example 18.

[0028] Figure 8 The average potentials of the calcium peroxide nanoparticles prepared in step (1) of Example 12, the drug-loaded calcium peroxide nanoparticles prepared in step (2) of Example 12, and the metal polyphenol network-encapsulated calcium peroxide nanoparticles prepared in Example 18 are as follows: A is the calcium peroxide nanoparticles prepared in step (1) of Example 12, B is the drug-loaded calcium peroxide nanoparticles prepared in step (2) of Example 12, and C is the metal polyphenol network-encapsulated calcium peroxide nanoparticles prepared in Example 18.

[0029] Figure 9 The particle size of the CK@PTF nanoparticles prepared in Example 19;

[0030] Figure 10 The particle size of the CK@PTC nanoparticles prepared in Example 20;

[0031] Figure 11 Inhibition rates of kaempferol (KAE), calcium peroxide nanoparticles (CaO2) prepared in step (1) of Example 12, CK nanoparticles prepared in step (2) of Example 12, and CK@PTM nanoparticles prepared in Example 18 on 4T1 cells at different drug administration concentrations;

[0032] Figure 12The effects of kaempferol (KAE) (4 μg / mL and 6 μg / mL) containing (+) calcium chelating agent and without (-) calcium chelating agent, calcium peroxide nanoparticles (CaO2) prepared in step (1) of Example 12, CK nanoparticles prepared in step (2) of Example 12 and CK@PTM nanoparticles prepared in Example 18 on the survival rate of 4T1 cells;

[0033] Figure 13 ROS content (20×) in 4T1 cells 12 h after administration of blank control group, kaempferol (KAE), CaO2 nanoparticles prepared in step (1) of Example 12, CK nanoparticles prepared in step (2) of Example 12, and CK@PTM nanoparticles prepared in Example 18;

[0034] Figure 14 The curves showing the changes in relative tumor volume in mice during treatment with blank control (Control), kaempferol (KAE), CaO2 nanoparticles prepared in step (1) of Example 12, CK nanoparticles prepared in step (2) of Example 12, and CK@PTM nanoparticles prepared in Example 18;

[0035] Figure 15 The results of mouse tumor pathological sections (10×, 100μm) after treatment with blank control group (Control), kaempferol (KAE), CaO2 nanoparticles prepared in step (1) of Example 12, CK nanoparticles prepared in step (2) of Example 12, and CK@PTM nanoparticles prepared in Example 18. Detailed Implementation

[0036] This invention provides a method for preparing calcium peroxide nanoparticles encapsulated in a metal polyphenol network, comprising the following steps:

[0037] (1) After mixing calcium peroxide nanoparticles, drugs and a first solvent, a chelation reaction was carried out to obtain drug-loaded calcium peroxide nanoparticles.

[0038] (2) After mixing the polymer solution and the polyphenol solution, a dehydration condensation reaction is carried out to obtain a polymer-modified polyphenol solution;

[0039] (3) The drug-loaded calcium peroxide nanoparticles obtained in step (1) are mixed with the second solvent, the polymer-modified polyphenol solution obtained in step (2) and the metal salt solution and then a coordination complexation reaction is carried out to obtain metal polyphenol network-encapsulated calcium peroxide nanoparticles.

[0040] The steps (1) and (2) are not in any particular order.

[0041] Unless otherwise specified, the present invention does not impose any special restrictions on the source of the raw materials, and commercially available products well known to those skilled in the art can be used.

[0042] In this invention, calcium peroxide nanoparticles, a drug, and a first solvent are mixed and then subjected to a chelation reaction to obtain drug-loaded calcium peroxide nanoparticles.

[0043] In this invention, the particle size of the calcium peroxide nanoparticles is preferably 90-160 nm.

[0044] In this invention, the preferred method for preparing the calcium peroxide nanoparticles is to mix calcium salt, stabilizer, third solvent, alkaline solution and hydrogen peroxide aqueous solution and then carry out a metathesis reaction to obtain calcium peroxide nanoparticles.

[0045] In this invention, the calcium salt is preferably a soluble calcium salt, and more preferably includes one or more of calcium chloride, calcium hypochlorite, calcium chlorate, calcium perchlorate, calcium bicarbonate, calcium nitrate, calcium hydrogen phosphate, calcium sulfate, calcium hydrogen sulfate, calcium bisulfite, calcium oxalate, calcium permanganate, calcium iodide, calcium bromide, calcium gluconate, and calcium lactate.

[0046] In this invention, the stabilizer is preferably a high molecular weight polymer, more preferably hyaluronic acid or polyvinylpyrrolidone (PVP), and even more preferably polyvinylpyrrolidone (PVP40). In this invention, the stabilizer can coordinate with calcium ions to form a protective layer, thereby affecting the growth and aggregation of nanocrystals. With the help of the stabilizer, spherical aggregates with uniform size and polycrystalline structure can be obtained.

[0047] In this invention, the mass ratio of the calcium salt to the stabilizer is preferably 1:(3-4), more preferably 1:3.5. By limiting the mass ratio of the calcium salt to the stabilizer within the above range, this invention can further obtain calcium peroxide nanoparticles with uniform size.

[0048] In this invention, the third solvent is preferably an alcohol solution, and more preferably anhydrous ethanol.

[0049] In this invention, the preferred mass ratio of the calcium salt to the volume ratio of the third solvent is (6-7) mg:1 mL, more preferably (6.5-7) mg:1 mL. By limiting the mass ratio of the calcium salt to the volume ratio of the third solvent within the above range, this invention facilitates the dissolution of the calcium salt.

[0050] In this invention, the concentration of the hydrogen peroxide aqueous solution is preferably 0.5 to 2 mol / L, more preferably 1 to 1.5 mol / L.

[0051] In this invention, the preferred ratio of the mass of the calcium salt to the molar amount of hydrogen peroxide in the hydrogen peroxide aqueous solution is (0.1–1) g: 0.8 mmol, more preferably (0.1–0.5) g: 0.8 mmol. By limiting the ratio of the mass of the calcium salt to the molar amount of hydrogen peroxide in the hydrogen peroxide aqueous solution to the above range, this invention is more conducive to the complete reaction of the two to form calcium peroxide nanoparticles.

[0052] In this invention, the alkaline solution preferably includes a weakly alkaline solution, and more preferably includes one or more of ammonia water, sodium bicarbonate aqueous solution and sodium carbonate aqueous solution.

[0053] In this invention, the preferred method for mixing the calcium salt, stabilizer, third solvent, alkaline solution, and hydrogen peroxide aqueous solution is as follows: the calcium salt, stabilizer, and third solvent are mixed to obtain a calcium salt solution, then an alkaline solution is added to obtain a reaction solution, and finally an aqueous hydrogen peroxide solution is added. In this invention, the pH value of the reaction solution is preferably 7-9, more preferably 8. This invention does not impose specific limitations on the concentration and amount of the alkaline solution, as long as the pH value of the reaction solution is within the above range.

[0054] In this invention, the hydrogen peroxide aqueous solution is preferably added dropwise. The present invention does not impose a particular limitation on the rate of addition; any rate well-known to those skilled in the art can be used. In this invention, the dropwise addition allows for a more complete reaction between hydrogen peroxide and calcium salt to form calcium peroxide nanoparticles. In this invention, the metathesis reaction begins immediately after the addition of the hydrogen peroxide aqueous solution.

[0055] In this invention, the temperature of the metathesis reaction is preferably 20–30°C; the time of the metathesis reaction is preferably 0.01–48 h, more preferably 5–36 h. In this invention, during the metathesis reaction, calcium salt and hydrogen peroxide react to form calcium peroxide nanoparticles. By limiting the temperature and time of the metathesis reaction within the above-mentioned ranges, this invention ensures that the calcium salt and hydrogen peroxide react fully.

[0056] After the metathesis reaction is completed, the products of the metathesis reaction are preferably centrifuged and washed sequentially to obtain calcium peroxide nanoparticles.

[0057] In this invention, the centrifugation rate is preferably 10,000 to 20,000 rpm, more preferably 12,000 to 18,000 rpm; the centrifugation time is preferably 5 to 60 min, more preferably 10 to 40 min.

[0058] In this invention, the solvent used for washing is preferably anhydrous ethanol. This invention does not impose any special limitations on the amount of solvent used or the number of washes; washing techniques well-known to those skilled in the art can be employed.

[0059] In this invention, the drug preferably includes chemotherapy drugs, and more preferably includes one or more of the following: kaempferol, gambogeylic acid, curcumin, elemol, tripterygium oleracea, oridonin, croton oil, artemisinin, baicalin, ginsenosides, gallic acid, resveratrol, matrine, astragalin, parisin, icariin, notoginseng saponin, ligustrazine, tanshinone, ursolic acid, cantharidin, ophiopogonin, platycodonin, apigenin, sinomenine, rosin, fraxin, bufotin, anthocyanin, rotenone, andrographolide, quercetin, kochiaside, osthol, paclitaxel, doxorubicin, camptothecin, fluorouracil, imatinib, cyclophosphamide, vincristine, mitoxantrone, bleomycin, nitrogen mustard hydrochloride, phenylalanine nitrogen mustard, etoposide, vinorelbine, methotrexate, and their derivatives.

[0060] In this invention, the first solvent is preferably an alcohol solvent, and more preferably anhydrous ethanol.

[0061] In this invention, the preferred method for mixing the calcium peroxide nanoparticles, the drug, and the first solvent is to: mix the calcium peroxide nanoparticles with a portion of the first solvent to obtain a calcium peroxide nanoparticle dispersion; mix the drug with the remaining first solvent to obtain a drug solution; and then mix the calcium peroxide nanoparticle dispersion with the drug solution.

[0062] In this invention, the concentration of the calcium peroxide nanoparticle dispersion is preferably 2-3 mg / mL, more preferably 2-2.5 mg / mL.

[0063] In this invention, the concentration of the drug solution is preferably 300–800 μg / mL, more preferably 500–700 μg / mL, and even more preferably 600 μg / mL. By limiting the concentrations of the calcium peroxide nanoparticle dispersion and the drug solution within the above ranges, this invention enables more complete dissolution or dispersion of the raw materials.

[0064] The present invention does not have a special limitation on the amount of the first solvent, as long as the concentration of the calcium peroxide nanoparticle dispersion and the drug solution are within the above range.

[0065] In this invention, the preferred volume ratio of the calcium peroxide nanoparticle dispersion to the drug solution is (3-15):1; the preferred mass ratio of the calcium peroxide nanoparticles to the drug is (2-60):1, more preferably (5-50):1. By limiting the volume ratio of the calcium peroxide nanoparticle dispersion to the drug solution and the mass ratio of the calcium peroxide nanoparticles to the drug within the above ranges, this invention can increase the drug loading capacity of the drug-loaded calcium peroxide nanoparticles, which is more beneficial for tumor treatment.

[0066] In this invention, the temperature of the chelation reaction is preferably 4–40°C, more preferably 20–40°C; the time of the chelation reaction is preferably 1–48 h, more preferably 12–48 h, and even more preferably 24–36 h. In this invention, during the chelation reaction, the drug binds to calcium peroxide through chelation. By limiting the temperature and time of the chelation reaction within the above-mentioned ranges, this invention can further increase the drug loading capacity of drug-loaded calcium peroxide nanoparticles.

[0067] After the chelation reaction is completed, the product of the chelation reaction is preferably centrifuged and washed sequentially to obtain drug-loaded calcium peroxide nanoparticles.

[0068] In this invention, the centrifugation rate is preferably 10,000 to 20,000 rpm, more preferably 12,000 to 18,000 rpm; the centrifugation time is preferably 5 to 60 min, more preferably 10 to 40 min.

[0069] In this invention, the solvent used for washing is preferably anhydrous ethanol. This invention does not impose any special limitations on the amount of solvent used or the number of washes; washing techniques well-known to those skilled in the art can be employed.

[0070] In this invention, a polymer solution and a polyphenol solution are mixed and then subjected to a dehydration condensation reaction to obtain a polymer-modified polyphenol solution.

[0071] In this invention, the solvent in the polymer solution is preferably water, more preferably ultrapure water; the polymer in the polymer solution is preferably aminated polyethylene glycol; and the concentration of the polymer solution is preferably 8–12 mg / mL, more preferably 10 mg / mL. By limiting the concentration of the polymer solution within the above range, this invention ensures complete dissolution.

[0072] In this invention, the solvent in the polyphenol solution is preferably a buffer salt solution, more preferably one of a phosphate buffer solution, a tris(hydroxymethyl)aminomethane buffer solution, a 3-morpholine propanesulfonic acid buffer solution, and a 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution. In this invention, the pH value of the buffer salt solution is preferably 7-10, more preferably 8-9. By using a buffer salt solution as a solvent and limiting its pH value within the above range, this invention enables the system to have a suitable pH value, which is more conducive to the reaction between the two.

[0073] In this invention, the concentration of the polyphenol solution is preferably 2-3 mmol / L, more preferably 2-2.5 mmol / L. Limiting the concentration of the polyphenol solution within this range ensures its complete dissolution.

[0074] In this invention, the polyphenols in the polyphenol solution preferably include curcumin, quercetin, puerarin, catechin, proanthocyanidins, luteolin, hesperidin, naringin, myricetin, rutin, pinocembrin, baicalin, gynostemma pentaphyllum, taxane, eugenol, apigenin, taxane, stigmosiderin, morin, emodin, juniperol, rutin, paeonol, kaempferol, sennaol, brown algae polyphenols, and polyflavanols. The following are one or more of the following: phenol, morin, polygalactoside, podophyllin, epinorcaryl aglycone, resveratrol, pterostilbene, tannic acid, salvianolic acid, chlorogenic acid, gallic acid, caffeic acid, ellagic acid, digalic acid, propyl gallate, epigallocatechin gallate, galloyl glucose, hydroquinone, epigallocatechin gallate, catechin gallate, gallocatechin gallate and their derivatives.

[0075] In this invention, the preferred mass ratio of the polymer in the polymer solution to the polyphenol in the polyphenol solution is (1-3):1, more preferably (1.5-2):1. This invention uses polymer-modified polyphenols to improve the dispersibility of the subsequently formed polyphenol network structure in water. By limiting the mass ratio of the polymer in the polymer solution to the polyphenol in the polyphenol solution to the above range, this invention further facilitates the dispersibility of the subsequently formed metal polyphenol network structure in water, thereby improving its tumor treatment efficacy.

[0076] In this invention, the temperature of the dehydration condensation reaction is preferably 4–40°C, more preferably 10–40°C, and even more preferably 20–40°C; the time of the dehydration condensation reaction is preferably 1–48 h, more preferably 1–36 h, and even more preferably 1–12 h. By limiting the temperature and time of the dehydration condensation reaction within the above ranges, this invention enables the polyphenols and polymers to react fully.

[0077] After obtaining drug-loaded calcium peroxide nanoparticles and polymer-modified polyphenol solution, the present invention mixes the drug-loaded calcium peroxide nanoparticles with a second solvent, the polymer-modified polyphenol solution and a metal salt solution and performs a coordination complexation reaction to obtain metal polyphenol network-encapsulated calcium peroxide nanoparticles.

[0078] In this invention, the second solvent is preferably an alcohol solvent, and more preferably anhydrous ethanol.

[0079] In this invention, the preferred mass ratio of the drug-loaded calcium peroxide nanoparticles to the volume ratio of the second solvent is (1-2) mg:1 mL, more preferably (1.5-2) mg:1 mL. By limiting the mass ratio of the drug-loaded calcium peroxide nanoparticles to the volume ratio of the second solvent within the above range, this invention ensures sufficient dispersion.

[0080] In this invention, the solvent in the metal salt solution is preferably a buffer salt solution, more preferably one of a phosphate buffer solution, a tris(hydroxymethyl)aminomethane buffer solution, a 3-morpholine propanesulfonic acid buffer solution, and a 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution. In this invention, the pH value of the buffer salt solution is preferably 7-10, more preferably 8-9. By using a buffer salt solution as the solvent and limiting its pH value within the above range, this invention enables the system to have a suitable pH value, which is more conducive to the coordination complexation reaction.

[0081] In this invention, the metal in the metal salt solution preferably includes one or more of Ag, Au, Al, Ba, Bi, Ca, Cd, Ce, Co, Cr, Cu, Eu, Fe, Fe, Gd, Mg, Mn, Mn, Mo, Ni, Pt, W, V, Zn, and Zr. This invention does not impose any particular limitation on the type of metal salt; any metal salt well-known to those skilled in the art can be used.

[0082] In this invention, the concentration of the metal salt solution is preferably 2 to 3 mmol / L, more preferably 2 to 2.5 mmol / L.

[0083] In this invention, the preferred molar ratio of polyphenols in the polyphenol solution to metal ions in the metal salt solution is (0.5–10):1, more preferably (2–8):1. By limiting the molar ratio of polyphenols in the polyphenol solution to metal ions in the metal salt solution to the above range, this invention enables the metal ions and polyphenols to undergo sufficient coordination and complexation reactions to form a network structure that encapsulates drug-loaded calcium peroxide nanoparticles, further improving their tumor treatment efficacy.

[0084] In this invention, the preferred molar ratio of calcium peroxide in the drug-loaded calcium peroxide nanoparticles to metal ions in the metal salt solution is (20-50):1, more preferably (25-40):1. By limiting the molar ratio of calcium peroxide in the drug-loaded calcium peroxide nanoparticles to metal ions in the metal salt solution to the above range, this invention enables the formed metal polyphenol network structure to more fully encapsulate the drug-loaded calcium peroxide nanoparticles, further improving its tumor treatment efficacy.

[0085] In this invention, the volume ratio of the polymer-modified polyphenol solution to the metal salt solution is preferably 1:(0.8-1.2), more preferably 1:1.

[0086] In this invention, the preferred method for mixing the drug-loaded calcium peroxide nanoparticles with the second solvent, the polymer-modified polyphenol solution, and the metal salt solution is as follows: the drug-loaded calcium peroxide nanoparticles are mixed with the second solvent to obtain a drug-loaded calcium peroxide nanoparticle dispersion, then the metal salt solution is added, followed by the addition of the polymer-modified polyphenol solution.

[0087] In this invention, the metal salt solution is preferably added slowly. There is no particular limitation on the rate of slow addition; a relatively slow rate is acceptable.

[0088] In this invention, the polymer-modified polyphenol solution is preferably added dropwise. This invention does not impose a particular limitation on the dropping rate; any dropping technique known to those skilled in the art can be used.

[0089] In this invention, the temperature of the coordination complexation reaction is preferably 4–40°C, more preferably 10–40°C, and even more preferably 20–40°C; the time of the coordination complexation reaction is preferably 1–48 h, more preferably 12–48 h, and even more preferably 24–36 h. In this invention, during the coordination complexation reaction, metal ions in the metal salt react with polyphenols to form a network structure that encapsulates drug-loaded calcium peroxide nanoparticles. By limiting the temperature and time of the coordination complexation reaction within the above-mentioned ranges, this invention enables the metal ions and polyphenols to fully react and encapsulate the drug-loaded calcium peroxide nanoparticles, further improving their tumor treatment efficacy.

[0090] After the coordination complexation reaction is completed, the present invention preferably centrifuges, washes and redisperses the product of the coordination complexation reaction in sequence.

[0091] In this invention, the centrifugation rate is preferably 5000-20000 rpm, more preferably 10000-15000 rpm; the centrifugation time is preferably 5-60 min, more preferably 10-40 min.

[0092] In this invention, the solvent used for washing is preferably ethanol. This invention does not impose any particular limitations on the amount of solvent used or the number of washes; any washing technique well-known to those skilled in the art can be used.

[0093] In this invention, the solvent used for redispersion is preferably ultrapure water.

[0094] This invention utilizes metal polyphenols to encapsulate calcium peroxide nanoparticles, protecting the unstable nanoparticles. The released metal ions catalyze the generation of hydroxyl radicals from hydrogen peroxide under acidic conditions, thereby killing tumor cells and achieving chemokinetic therapy. Furthermore, it consumes glutathione, enhancing the accumulation of reactive oxygen species and thus strengthening the anti-tumor effect. When dispersed in an acidic environment, the metal polyphenol network layer rapidly degrades, exposing the calcium peroxide nanoparticles and explosively releasing large amounts of calcium ions and oxygen, more effectively activating calcium overload and alleviating the hypoxic tumor microenvironment. By loading drugs, it improves the effective accumulation of drugs at the tumor site, achieving combined chemokinetic therapy and chemotherapy, thereby activating the anti-tumor immune response and improving the anti-tumor effect.

[0095] The present invention provides a metal polyphenol network-encapsulated calcium peroxide nanoparticle prepared by the preparation method described above, comprising drug-loaded calcium peroxide nanoparticles and a metal polyphenol network encapsulating the surface of the drug-loaded calcium peroxide nanoparticles.

[0096] The metal polyphenol network-encapsulated calcium peroxide nanoparticles prepared in this invention have better tumor treatment effects.

[0097] The present invention also provides the application of the metal polyphenol network-encapsulated calcium peroxide nanoparticles described in the above technical solution in the preparation of tumor drugs.

[0098] This invention does not impose any particular limitation on the technical solution for the application of the metal polyphenol network-encapsulated calcium peroxide nanoparticles in the preparation of tumor drugs. Any technical solution well known to those skilled in the art for the application of metal polyphenol network-encapsulated calcium peroxide nanoparticles in the preparation of tumor drugs can be used.

[0099] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0100] Example 1

[0101] (1) Dissolve 0.4 g of calcium chloride and 1.4 g of polyvinylpyrrolidone (PVP40) in 60 mL of anhydrous ethanol (the mass ratio of calcium chloride to the volume ratio of anhydrous ethanol is 6.7 mg: 1 mL). Then, add 4 mL of 0.8 mol / L ammonia water at 25 °C with stirring until the pH of the solution is 8. Then, add 0.8 mL of 1 mol / L hydrogen peroxide aqueous solution (the mass ratio of calcium chloride to PVP40 is 1:3.5, and the mass ratio of calcium chloride to the molar amount of hydrogen peroxide in the hydrogen peroxide aqueous solution is 0.4 g: 0.8 mmol). After the addition is complete, stir for 2 min. After the reaction is complete, centrifuge at 15000 rpm for 20 min, collect the calcium peroxide nanoparticles, and wash them with ethanol 5 times to obtain calcium peroxide nanoparticles, which are denoted as CaO2 nanoparticles.

[0102] (2) The calcium peroxide nanoparticles obtained in step (1) were mixed with 15 mL of anhydrous ethanol to obtain a calcium peroxide nanoparticle dispersion with a concentration of 2.14 mg / mL; Kaempferol (KAE) was mixed with anhydrous ethanol to obtain a kaempferol solution with a concentration of 600 μg / mL; the calcium peroxide nanoparticle dispersion and the kaempferol solution were mixed at a volume ratio of 15:1 (the mass ratio of calcium peroxide nanoparticles to kaempferol was 53.5:1), and a chelation reaction was carried out at 25 °C for 24 h. After centrifugation at 15000 rpm for 20 min, the calcium peroxide nanoparticles loaded with kaempferol were collected and denoted as CK nanoparticles.

[0103] Example 2

[0104] The volume ratio of calcium peroxide nanoparticle dispersion to kaempferol solution in step (2) of Example 1 is replaced with 12:1. At this time, the mass ratio of calcium peroxide nanoparticles to kaempferol is 42.8:1. All other parameters are the same as in Example 1.

[0105] Example 3

[0106] The volume ratio of calcium peroxide nanoparticle dispersion to kaempferol solution in step (2) of Example 1 is replaced with 10:1. At this time, the mass ratio of calcium peroxide nanoparticles to kaempferol is 35.7:1. All other parameters are the same as in Example 1.

[0107] Example 4

[0108] Replace the volume ratio of calcium peroxide nanoparticle dispersion to kaempferol solution in step (2) of Example 1 with 7:1. At this time, the mass ratio of calcium peroxide nanoparticles to kaempferol is 25:1. All other parameters are the same as in Example 1.

[0109] Example 5

[0110] The volume ratio of calcium peroxide nanoparticle dispersion to kaempferol solution in step (2) of Example 1 is replaced with 5:1. At this time, the mass ratio of calcium peroxide nanoparticles to kaempferol is 17.8:1. All other parameters are the same as in Example 1.

[0111] Example 6

[0112] The volume ratio of calcium peroxide nanoparticle dispersion to kaempferol solution in step (2) of Example 1 is replaced with 3:1. At this time, the mass ratio of calcium peroxide nanoparticles to kaempferol is 10.7:1. All other parameters are the same as in Example 1.

[0113] The particle size of the CaO2 nanoparticles prepared in step (1) of Example 1 and the drug-loaded calcium peroxide nanoparticles prepared in Examples 1-6 were measured, and the results are as follows: Figure 1 As shown. The encapsulation efficiency and drug loading of the drug-loaded calcium peroxide nanoparticles prepared in Examples 1-6 were measured, and the results are as follows. Figure 2As shown, the specific test steps for encapsulation efficiency and drug loading are as follows: After the chelation reaction in Examples 1-6 is completed, the system is centrifuged at 14000 rpm at 4°C for 20 min. The supernatant is collected, and the centrifuged nanoparticles are washed twice with ethanol. After centrifugation at 14000 rpm at 4°C for 20 min, the supernatant is collected. The three supernatants are combined, and the absorbance is measured at the maximum absorption wavelength. The free content is calculated based on the linear curve and denoted as W. 游 The initial amount of kaempferol added is denoted as W. 总 The total amount of CK nanoparticles is denoted as W, and its encapsulation efficiency is calculated using the formula: Encapsulation efficiency (%) = [(W)] 总- W 游 ) / W 总 ]×100%, calculate its drug loading using the formula: Drug loading (%) = [(W 总- W 游 [) / W]×100%, each example was measured 3 times, and the average value was taken. From Figure 1 and Figure 2 As can be seen, as the volume ratio of calcium peroxide nanoparticle dispersion to kaempferol solution decreases, i.e., the mass ratio of calcium peroxide nanoparticles to kaempferol decreases, the particle size of CK nanoparticles does not change significantly, the encapsulation efficiency gradually decreases, and the drug loading gradually increases.

[0114] Example 7

[0115] (1) Dissolve 0.4 g calcium chloride and 1.4 g polyvinylpyrrolidone (PVP40) in 60 mL of anhydrous ethanol (the mass ratio of calcium chloride to the volume ratio of anhydrous ethanol is 6.7 mg: 1 mL). Then, add 4 mL of 0.8 mol / L ammonia water at 25 °C with stirring until the pH of the solution is 8. Then, add 0.8 mL of 1 mol / L hydrogen peroxide aqueous solution (the mass ratio of calcium chloride to PVP40 is 1:3.5, and the mass ratio of calcium chloride to the molar amount of hydrogen peroxide in the hydrogen peroxide aqueous solution is 0.4 g: 0.8 mmol). After the addition is complete, stir for 2 min. After the reaction is complete, centrifuge at 15000 rpm for 20 min, collect the calcium peroxide nanoparticles, and wash them with ethanol 5 times to obtain calcium peroxide nanoparticles, which are denoted as CaO2 nanoparticles.

[0116] (2) The calcium peroxide nanoparticles obtained in step (1) were mixed with 1.5 mL of anhydrous ethanol to obtain a calcium peroxide nanoparticle dispersion with a concentration of 2.14 mg / mL; Kaempferol (KAE) was mixed with anhydrous ethanol to obtain a kaempferol solution with a concentration of 300 μg / mL; 1.5 mL of calcium peroxide nanoparticle dispersion and 300 μL of kaempferol solution were mixed (the mass ratio of calcium peroxide nanoparticles to kaempferol was 35.7:1), and the mixture was subjected to a chelation reaction at 25 °C for 24 h. After centrifugation at 15000 rpm for 20 min, the calcium peroxide nanoparticles loaded with kaempferol were collected and denoted as CK nanoparticles.

[0117] Example 8

[0118] Replace the concentration of the kaempferol solution in step (2) of Example 7 with 400 μg / mL. At this time, the mass ratio of calcium peroxide nanoparticles to kaempferol is 26.75:1, and other parameters are the same as in Example 7.

[0119] Example 9

[0120] Replace the concentration of kaempferol solution in step (2) of Example 7 with 500 μg / mL. At this time, the mass ratio of calcium peroxide nanoparticles to kaempferol is 21.4:1. All other parameters are the same as in Example 7.

[0121] Example 10

[0122] Replace the concentration of the kaempferol solution in step (2) of Example 7 with 700 μg / mL. At this time, the mass ratio of calcium peroxide nanoparticles to kaempferol is 15.29:1, and other parameters are the same as in Example 7.

[0123] Example 11

[0124] Replace the concentration of the kaempferol solution in step (2) of Example 7 with 800 μg / mL. At this time, the mass ratio of calcium peroxide nanoparticles to kaempferol is 13.375:1, and other parameters are the same as in Example 7.

[0125] Using the same testing method, the particle size of the CaO2 nanoparticles prepared in step (1) of Example 7, the drug-loaded calcium peroxide nanoparticles prepared in Example 5, and the drug-loaded calcium peroxide nanoparticles prepared in Examples 7-11 were measured, and the results are as follows: Figure 3 As shown. The encapsulation efficiency and drug loading of the drug-loaded calcium peroxide nanoparticles prepared in Example 5 and Examples 7-11 were measured, and the results are as follows. Figure 4 As shown. From Figure 3 and Figure 4 As can be seen, with the increase of KAE concentration, the particle size of CK nanoparticles did not change significantly, the encapsulation efficiency gradually decreased, and the drug loading gradually increased.

[0126] Example 12

[0127] (1) Dissolve 0.4 g calcium chloride and 1.4 g polyvinylpyrrolidone (PVP40) in 60 mL of anhydrous ethanol (the mass ratio of calcium chloride to the volume ratio of anhydrous ethanol is 6.7 mg: 1 mL). Then, add 4 mL of 0.8 mol / L ammonia water at 25 °C with stirring until the pH of the solution is 8. Then, add 0.8 mL of 1 mol / L hydrogen peroxide aqueous solution (the mass ratio of calcium chloride to PVP40 is 1:3.5, and the mass ratio of calcium chloride to the molar amount of hydrogen peroxide in the hydrogen peroxide aqueous solution is 0.4 g: 0.8 mmol). After the addition is complete, stir for 2 min. After the reaction is complete, centrifuge at 15000 rpm for 20 min, collect the calcium peroxide nanoparticles, and wash them with ethanol 5 times to obtain calcium peroxide nanoparticles, which are denoted as CaO2 nanoparticles.

[0128] (2) The calcium peroxide nanoparticles obtained in step (1) were mixed with 15 mL of anhydrous ethanol to obtain a calcium peroxide nanoparticle dispersion with a concentration of 2.14 mg / mL; Kaempferol (KAE) was mixed with anhydrous ethanol to obtain a kaempferol solution with a concentration of 600 μg / mL; the calcium peroxide nanoparticle dispersion and the kaempferol solution were mixed at a volume ratio of 5:1 (the mass ratio of calcium peroxide nanoparticles to kaempferol was 17.8:1), and a chelation reaction was carried out at 25 °C for 24 h. After centrifugation at 15000 rpm for 20 min, the calcium peroxide nanoparticles loaded with kaempferol were collected and denoted as CK nanoparticles.

[0129] (3) Add 2 mL of 10 mg / mL aminated polyethylene glycol aqueous solution to 3 mL of 3-morpholine propanesulfonic acid buffer solution containing 2.4 mmol / L tannic acid (relative molecular mass of 1701.2, Maclean CAS No.: 1401-55-4, catalog number: T818845) (the pH value of the buffer solution is 8, and the mass ratio of aminated polyethylene glycol to tannic acid is 5:3). React at 25℃ for 1 h to obtain polyethylene glycol modified tannic acid solution, denoted as PEG-TA solution;

[0130] (4) The calcium peroxide nanoparticles loaded with kaempferol prepared in step (2) were mixed with 18 mL of anhydrous ethanol (the mass ratio of the calcium peroxide nanoparticles loaded with kaempferol to the volume ratio of anhydrous ethanol was 1.68 mg: 1 mL) to obtain a drug-loaded calcium peroxide nanoparticle dispersion with a concentration of 1.68 mg / mL. 0.4 mL of 3-morpholinopropanesulfonic acid buffer solution containing 2.4 mmol / L manganese chloride (pH value of the buffer solution was 8) was slowly added, and then an equal volume (0.4 mL) of PEG-T obtained in step (3) was added dropwise. Solution A (the volume ratio of PEG-TA solution to 3-morpholinopropanesulfonic acid buffer solution containing manganese chloride is 1:1, the molar ratio of tannic acid to manganese ions is 5:8, and the molar ratio of calcium peroxide to manganese ions in calcium peroxide nanoparticles loaded with kaempferol is 46:1) was stirred at 25°C for 24 h, centrifuged at 10000 rpm for 10 min, washed twice with ethanol, and then dispersed in 1.5 mL of ultrapure water to obtain metal polyphenol network-encapsulated calcium peroxide nanoparticles, denoted as CK@PTM nanoparticles.

[0131] Example 13

[0132] Replace the volume of the 3-morpholinopropanesulfonic acid buffer solution containing 2.4 mmol / L manganese chloride in step (4) of Example 12 with 0.45 mL, and replace the volume of the PEG-TA solution with 0.45 mL. At this time, the molar ratio of tannic acid to manganese ions is 5:8, and the molar ratio of calcium peroxide to manganese ions in the calcium peroxide nanoparticles loaded with kaempferol is 41:1. All other parameters are the same as in Example 12.

[0133] Example 14

[0134] Replace the volume of the 3-morpholinopropanesulfonic acid buffer solution containing 2.4 mmol / L manganese chloride in step (4) of Example 12 with 0.5 mL, and replace the volume of the PEG-TA solution with 0.5 mL. At this time, the molar ratio of tannic acid to manganese ions is 5:8, and the molar ratio of calcium peroxide to manganese ions in the calcium peroxide nanoparticles loaded with kaempferol is 37:1. All other parameters are the same as in Example 12.

[0135] Example 15

[0136] Replace the volume of the 3-morpholinopropanesulfonic acid buffer solution containing 2.4 mmol / L manganese chloride in step (4) of Example 12 with 0.55 mL, and replace the volume of the PEG-TA solution with 0.55 mL. At this time, the molar ratio of tannic acid to manganese ions is 5:8, and the molar ratio of calcium peroxide to manganese ions in the calcium peroxide nanoparticles loaded with kaempferol is 34:1. All other parameters are the same as in Example 12.

[0137] Example 16

[0138] Replace the volume of the 3-morpholinopropanesulfonic acid buffer solution containing 2.4 mmol / L manganese chloride in step (4) of Example 12 with 0.6 mL, and replace the volume of the PEG-TA solution with 0.6 mL. At this time, the molar ratio of tannic acid to manganese ions is 5:8, and the molar ratio of calcium peroxide to manganese ions in the calcium peroxide nanoparticles loaded with kaempferol is 31:1. All other parameters are the same as in Example 12.

[0139] Example 17

[0140] In step (4) of Example 12, the volume of the 3-morpholinopropanesulfonic acid buffer solution containing 2.4 mmol / L manganese chloride was replaced with 0.65 mL, and the volume of the PEG-TA solution was replaced with 0.65 mL. At this time, the molar ratio of tannic acid to manganese ions was 5:8, and the molar ratio of calcium peroxide to manganese ions in the calcium peroxide nanoparticles loaded with kaempferol was 29:1. All other parameters were the same as in Example 12.

[0141] Example 18

[0142] Replace the volume of the 3-morpholinopropanesulfonic acid buffer solution containing 2.4 mmol / L manganese chloride in step (4) of Example 12 with 0.7 mL, and replace the volume of the PEG-TA solution with 0.7 mL. At this time, the molar ratio of tannic acid to manganese ions is 5:8, and the molar ratio of calcium peroxide to manganese ions in the calcium peroxide nanoparticles loaded with kaempferol is 26:1. All other parameters are the same as in Example 12.

[0143] The particle size of the metal polyphenol network-encapsulated calcium peroxide nanoparticles prepared in Examples 12-18 was tested using the same testing method, and the results are as follows: Figure 5 As shown. From Figure 5 As can be seen, the particle size of CK@PTM nanoparticles gradually increases with the gradual increase in the volume of added manganese chloride and tannic acid.

[0144] Macroscopic images of the calcium peroxide nanoparticle dispersion prepared in Example 12, the drug-loaded calcium peroxide nanoparticle dispersion prepared in Example 12, and the metal polyphenol network-encapsulated calcium peroxide nanoparticle dispersion prepared in Example 18 are shown below. Figure 6 As shown, from left to right, the images depict a calcium peroxide nanoparticle dispersion, a drug-loaded calcium peroxide nanoparticle dispersion, and a metal polyphenol network-encapsulated calcium peroxide nanoparticle dispersion. Figure 6 As can be seen, the appearance colors of the calcium peroxide nanoparticle dispersion, the drug-loaded calcium peroxide nanoparticle dispersion, and the metal polyphenol network-encapsulated calcium peroxide nanoparticle dispersion are light blue, yellow, and brown, respectively. The changes in their appearance colors indirectly verify the successful synthesis of CK@PTM nanoparticles.

[0145] The particle sizes of the calcium peroxide nanoparticles prepared in step (1) of Example 12, the drug-loaded calcium peroxide nanoparticles prepared in step (2) of Example 12, and the metal polyphenol network-encapsulated calcium peroxide nanoparticles prepared in Example 18 were measured, and the results are as follows: Figure 7 As shown, A represents the calcium peroxide nanoparticles prepared in step (1) of Example 12, B represents the drug-loaded calcium peroxide nanoparticles prepared in step (2) of Example 12, and C represents the metal polyphenol network-encapsulated calcium peroxide nanoparticles prepared in Example 18. Figure 7 As can be seen, the average particle sizes of CaO2, CK, and CK@PTM nanoparticles are 114.9±2.62 nm, 101.3±0.74 nm, and 138.0±1.07 nm, respectively.

[0146] The average potentials of the calcium peroxide nanoparticles prepared in step (1) of Example 12, the drug-loaded calcium peroxide nanoparticles prepared in step (2) of Example 12, and the metal polyphenol network-encapsulated calcium peroxide nanoparticles prepared in Example 18 were tested, and the results are as follows: Figure 8 As shown, A represents the calcium peroxide nanoparticles prepared in step (1) of Example 12, B represents the drug-loaded calcium peroxide nanoparticles prepared in step (2) of Example 12, and C represents the metal polyphenol network-encapsulated calcium peroxide nanoparticles prepared in Example 18. Figure 8 As can be seen, the average potentials of CaO2, CK, and CK@PTM nanoparticles are 1.63±0.19mV, -31.6±0.19mV, and -11.5±2.83mV, respectively.

[0147] Example 19

[0148] (1) Dissolve 0.4 g of calcium chloride and 1.4 g of polyvinylpyrrolidone (PVP40) in 60 mL of anhydrous ethanol (the mass ratio of calcium chloride to the volume ratio of anhydrous ethanol is 6.7 mg: 1 mL). Then, add 4 mL of 0.8 mol / L ammonia water at 25 °C with stirring until the pH of the solution is 8. Then, add 0.8 mL of 1 mol / L hydrogen peroxide aqueous solution (the mass ratio of calcium chloride to PVP40 is 1:3.5, and the mass ratio of calcium chloride to the molar amount of hydrogen peroxide in the hydrogen peroxide aqueous solution is 0.4 g: 0.8 mmol). After the addition is complete, stir for 2 min. After the reaction is complete, centrifuge at 15000 rpm for 20 min, collect the calcium peroxide nanoparticles, and wash them with ethanol 5 times to obtain calcium peroxide nanoparticles, which are denoted as CaO2 nanoparticles.

[0149] (2) The calcium peroxide nanoparticles obtained in step (1) were mixed with 15 mL of anhydrous ethanol to obtain a calcium peroxide nanoparticle dispersion with a concentration of 2.14 mg / mL; Kaempferol (KAE) was mixed with anhydrous ethanol to obtain a kaempferol solution with a concentration of 600 μg / mL; the calcium peroxide nanoparticle dispersion and the kaempferol solution were mixed at a volume ratio of 5:1 (the mass ratio of calcium peroxide nanoparticles to kaempferol was 17.8:1), and a chelation reaction was carried out at 25 °C for 24 h. After centrifugation at 15000 rpm for 20 min, the calcium peroxide nanoparticles loaded with kaempferol were collected and denoted as CK nanoparticles.

[0150] (3) Add 2 mL of 10 mg / mL aminated polyethylene glycol aqueous solution to 3 mL of 3-morpholinopropanesulfonic acid buffer solution containing 2.4 mmol / L tannic acid (the pH value of the buffer solution is 8, and the mass ratio of aminated polyethylene glycol to tannic acid is 5:3), react at 25 °C for 1 h to obtain polyethylene glycol modified tannic acid solution, denoted as PEG-TA solution;

[0151] (4) The calcium peroxide nanoparticles loaded with kaempferol prepared in step (2) were mixed with 18 mL of anhydrous ethanol (the mass ratio of the calcium peroxide nanoparticles loaded with kaempferol to the volume ratio of anhydrous ethanol was 1.68 mg: 1 mL) to obtain a drug-loaded calcium peroxide nanoparticle dispersion with a concentration of 1.68 mg / mL. 0.7 mL of 3-morpholinopropanesulfonic acid buffer solution containing 2.4 mmol / L ferric chloride (pH value of the buffer solution was 8) was slowly added, and then an equal volume of the PEG-TA solution obtained in step (3) was added dropwise. The volume ratio of the PEG-TA solution to the total volume of the 3-morpholinopropanesulfonic acid buffer solution containing ferric chloride was 1:1, the molar ratio of tannic acid to iron ions was 5:8, and the molar ratio of calcium peroxide to iron ions in the calcium peroxide nanoparticles loaded with kaempferol was 26:1. The reaction was stirred at 25°C for 24 h, centrifuged at 10000 rpm for 10 min, washed twice with ethanol, and then dispersed in 1.5 mL of ultrapure water to obtain metal polyphenol network-encapsulated calcium peroxide nanoparticles, denoted as CK@PTF nanoparticles.

[0152] The particle size of the CK@PTF nanoparticles obtained in Example 19 was measured three times, and the results are as follows: Figure 9 As shown. From Figure 9 As can be seen, the particle size and PDI of CK@PTF nanoparticles are 156.5 nm and 0.135, respectively.

[0153] Example 20

[0154] The 3-morpholinopropanesulfonic acid buffer solution containing ferric chloride in step (4) of Example 19 was replaced with a 3-morpholinopropanesulfonic acid buffer solution containing copper chloride. All other parameters were the same as in Example 19. Metal polyphenol network-encapsulated calcium peroxide nanoparticles were obtained and denoted as CK@PTC nanoparticles.

[0155] The particle size of the CK@PTC nanoparticles obtained in Example 20 was measured three times, and the results are as follows: Figure 10 As shown. From Figure 10 As can be seen, the particle size and PDI of CK@PTC nanoparticles are 199.8 nm and 0.207, respectively.

[0156] Application Example 1

[0157] The in vitro antitumor effects of kaempferol, calcium peroxide nanoparticles prepared in step (1) of Example 12, CK nanoparticles prepared in step (2) of Example 12, and CK@PTM nanoparticles prepared in Example 18 were tested respectively: Logarithmic growth phase breast cancer 4T1 cells were taken and subjected to 2.5×10 4 Cells were seeded at a density of 100 cells / mL in 96-well cell culture plates and cultured at 37°C in a 5% CO2 incubator. When the cell density reached 75%, the old culture medium was discarded, and different concentrations (2, 4, 6, 8, 10 μg / mL) of fresh culture medium containing kaempferol (KAE), CaO2 nanoparticles prepared in step (1) of Example 12, CK nanoparticles prepared in step (2) of Example 12, and CK@PTM nanoparticles prepared in Example 18 were added to each experimental group, with three replicates per group. The 96-well cell culture plates were then cultured for another 24 hours. The old culture medium was discarded, and 100 μL of medium containing 10% CCK-8 was added to each well. After incubation for 2 hours, the plates were removed, and the absorbance at 450 nm was measured using a full-wavelength microplate reader. The cell inhibition rate was calculated, and the results are as follows: Figure 11 As shown. From Figure 11 As can be seen, the cytotoxicity of all experimental groups against 4T1 cells showed a significant concentration-dependent effect. When the drug concentration was 10 μg / mL, the inhibition rates of kaempferol (KAE), CaO2 nanoparticles, CK nanoparticles, and CK@PTM nanoparticles against 4T1 cells were 15.46±3.91%, 64.89±4.00%, 83.01±1.55%, and 90.61±0.94%, respectively, indicating that CK@PTM nanoparticles have a good anti-tumor effect in vitro.

[0158] Log-phase breast cancer 4T1 cells were harvested and processed at a concentration of 2.5 × 10⁻⁶. 4Cells were seeded at a density of 100 cells / mL in 96-well cell culture plates and cultured at 37°C in a 5% CO2 incubator. When the cell density reached 75%, the old culture medium was discarded. Fresh culture medium was added to the experimental groups: one without calcium chelating agent and the other containing kaempferol (KAE) with 4 μg / mL calcium chelating agent (KAE concentrations were 4 μg / mL and 6 μg / mL, respectively). CaO2 nanoparticles prepared in step (1) of Example 12, CK nanoparticles prepared in step (2) of Example 12, and CK@PTM nanoparticles prepared in Example 18. Three replicates were set for each group. The 96-well cell culture plates were then cultured for another 24 hours. The old culture medium was discarded, and 100 μL of medium containing 10% CCK-8 was added to each well. After incubation for 2 hours, the absorbance of each well at 450 nm was measured using a full-wavelength microplate reader, and the cell inhibition rate was calculated. The results are as follows: Figure 12 As shown. From Figure 12 As can be seen from the data, after the addition of calcium chelating agents, the cell survival rate of all groups except the blank control group and the kaempferol group was improved, indicating that calcium overload is one of the reasons for the induced death of 4T1 cells by CaO2 nanoparticles, CK nanoparticles and CK@PTM nanoparticles.

[0159] 4T1 cells in logarithmic growth phase were harvested and treated at a rate of 3 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 10 cells / mL in 48-well plates and cultured in an incubator. When the cells reached 75% confluence, the old culture medium was removed, and fresh culture medium containing different concentrations (4 μg / mL, 6 μg / mL) of kaempferol (KAE), CaO2 nanoparticles prepared in step (1) of Example 12, CK nanoparticles prepared in step (2) of Example 12, and CK@PTM nanoparticles prepared in Example 18 was added, with 3 replicates for each group. After incubation in an incubator for 12 h, the culture medium was removed, and DCFH-DA (10 μM) staining solution was added to each well. After incubation at 37°C for 20 min, the fluorescence signal was observed under a fluorescence microscope, images were acquired and analyzed, and the results are as follows: Figure 13 As shown. From Figure 13 As can be seen, almost no green fluorescence was observed in the control group, while the kaempferol group showed a weaker green fluorescence signal, indicating that kaempferol can promote the production of ROS in 4T1 cells. Due to the dual effects of CaO2 nanoparticles and kaempferol, the fluorescence signal of the CK nanoparticle group was slightly greater than that of the CaO2 nanoparticle group, and the CK@PTM nanoparticle group showed the strongest fluorescence signal. This can be attributed to the triple effect of CaO2, kaempferol, and the metal polyphenol network.

[0160] Application Example 2

[0161] The in vivo antitumor effects of kaempferol, calcium peroxide nanoparticles prepared in step (1) of Example 12, CK nanoparticles prepared in step (2) of Example 12, and CK@PTM nanoparticles prepared in Example 18 were tested respectively: 4T1 cells in logarithmic growth phase were used, and 0.1 mL of cells with a concentration of 1.5 × 10⁻⁶ were added. 7 A cell suspension of 10 cells / mL was inoculated into the fat pad of the fourth nipple on the right side of mice. After inoculation, tumor growth was observed daily, and the major axis (a, in mm) and minor axis (b, in mm) of the mouse tumor were measured and recorded using calipers. The tumor volume was calculated using the formula V = a × b. 2 / 2, calculate the tumor volume in the mouse, and wait until the tumor volume reaches 100 mm. 3 When the tumor-bearing mice were successfully modeled, they were randomly divided into 5 groups. Each group was injected with saline, kaempferol (KAE), calcium peroxide nanoparticles prepared in step (1) of Example 12, CK nanoparticles prepared in step (2) of Example 12, and CK@PTM nanoparticles prepared in Example 18 via tail vein injection. The drugs were administered every 2 days at a dose of 2 mg / kg for a total of 7 administrations. Before each administration, the tumor volume of each group was measured and calculated using calipers, and a curve showing the change in tumor volume during treatment was plotted. After treatment, the mice were sacrificed, tumor tissue was collected, photographed, and weighed. The tumor size and body weight of different groups were compared to calculate the tumor inhibition rate. Subsequently, the tumor tissue was embedded in paraffin, and the apoptosis of tumor cells was detected by HE staining, TUNEL staining, and Ki67 staining. The in vivo antitumor effect of CK@PTM nanoparticles was investigated, and the results were as follows: Figure 14 and Figure 15 As shown. From Figure 14 As can be seen, the relative tumor volume of mice in all groups showed an increasing trend, but the growth rate differed. The saline (Control) group showed the most rapid growth, followed by the kaempferol (KAE) group. There was no significant difference in growth rate between the CaO2 nanoparticle group and the CK nanoparticle group. The CK@PTM group showed the slowest growth, indicating that CK@PTM nanoparticles have a good inhibitory effect on tumor growth. Figure 15The results of HE staining showed that the tumor cells in the saline (Control) group had a high density, intact cell morphology, and good growth status. The kaempferol (KAE) group showed some degree of nuclear shrinkage and karyolysis. The CaO2 nanoparticle group, CK nanoparticle group, and CK@PTM nanoparticle group all showed large-area karyolysis and atrophy, with the CK@PTM nanoparticle group showing the most severe tumor tissue damage. Ki67 staining results showed that the saline (Control) group contained a large number of proliferating tumor cells, accounting for 63.30±4.30%. The proportion of brownish cells in the kaempferol (KAE) group was significantly reduced, with a small number of proliferating tumor cells present. The proportion of proliferating tumor cells was 36.63±2.07%; almost no proliferating cells were found in the CaO2 nanoparticle group, CK nanoparticle group, and CK@PTM nanoparticle group, indicating that CK@PTM nanoparticles can significantly inhibit tumor cell proliferation. TUNEL staining results showed that only a small number of apoptotic tumor cells were present in the saline (Control) group. Compared with the control group, the number of apoptotic tumor cells was significantly increased in the kaempferol (KAE) group. A large number of apoptotic tumor cells were present in the CaO2 nanoparticle group, CK nanoparticle group, and CK@PTM nanoparticle group, accounting for 81.77±3.82%, which was consistent with the results of HE staining and TUNEL staining. These results indicate that CK@PTM nanoparticles can significantly inhibit tumor cell proliferation and have a strong tumor-killing effect.

[0162] In summary, the metal polyphenol network-encapsulated calcium peroxide nanoparticles prepared in this invention achieve a combined treatment of chemokine therapy and chemotherapy, resulting in better anti-tumor effects.

[0163] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing calcium peroxide nanoparticles encapsulated in a metal polyphenol network, comprising the following steps: (1) After mixing calcium peroxide nanoparticles, drugs and a first solvent, a chelation reaction was carried out to obtain drug-loaded calcium peroxide nanoparticles; The mass ratio of the calcium peroxide nanoparticles to the drug is (10~55):1; the drug includes kaempferol. (2) The polymer solution and the polyphenol solution are mixed and then subjected to a dehydration condensation reaction to obtain a polymer-modified polyphenol solution; the polymer in the polymer solution is aminated polyethylene glycol; the mass ratio of the polymer in the polymer solution to the polyphenol in the polyphenol solution is (1~3):1; the polyphenol in the polyphenol solution includes tannic acid; (3) The drug-loaded calcium peroxide nanoparticles obtained in step (1) are mixed with the second solvent, the polymer-modified polyphenol solution obtained in step (2) and the metal salt solution and then a coordination complexation reaction is carried out to obtain metal polyphenol network-encapsulated calcium peroxide nanoparticles. The metal in the metal salt solution is Mn; the molar ratio of polyphenol in the polyphenol solution of step (2) to metal ions in the metal salt solution of step (3) is (0.5~10):1; the molar ratio of calcium peroxide in the drug-loaded calcium peroxide nanoparticles to metal ions in the metal salt solution is (20~50):

1. The steps (1) and (2) are not in any particular order.

2. The preparation method according to claim 1, characterized in that, The chelation reaction temperature in step (1) is 4~40℃, and the chelation reaction time is 1~48h.

3. The preparation method according to claim 1, characterized in that, The temperature of the dehydration condensation reaction in step (2) is 4~40℃, and the time of the dehydration condensation reaction is 1~48h.

4. The preparation method according to claim 1, characterized in that, The temperature of the coordination complexation reaction in step (3) is 4~40℃, and the time of the coordination complexation reaction is 1~48h.

5. The metal polyphenol network-encapsulated calcium peroxide nanoparticles prepared by the preparation method according to any one of claims 1 to 4, comprising drug-loaded calcium peroxide nanoparticles and a metal polyphenol network encapsulating the surface of the drug-loaded calcium peroxide nanoparticles.

6. The use of the metal polyphenol network-encapsulated calcium peroxide nanoparticles according to claim 5 in the preparation of drugs for treating breast cancer.

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