A GSH / pH dual-responsive Cu2O@Cu 2+ / ZIF-67@ZnPc core-shell structure nanodrug

Abstract

The application discloses a GSH / pH dual-response Cu2O@Cu 2+ / ZIF-67@ZnPc core-shell structure nano drug, a preparation method and application thereof, and belongs to the technical field of metal organic framework materials as drug transportation carriers. First, Cu2O nanospheres are used as templates, sodium dodecyl sulfonate is used to change the potential of the Cu2O nanospheres, and then the Cu2O nanospheres are reacted with a cobalt nitrate hexahydrate solution and a 2-methyl imidazole methanol solution to obtain Cu2O@Cu 2+ / ZIF-67 core-shell structure nano material, and then a photosensitizer, a tetra-carboxy phthalocyanine zinc and a polypropylene glycol and ethylene oxide polyadduct are added to obtain the target nano drug. The nano drug is based on the synthesis of a uniform doped core-shell MOFs structure from a Cu2O self-template, has good product stability and high drug loading rate, and can activate chemical kinetics and photodynamic synergy treatment (CDT / PDT) in a tumor microenvironment.

Description

Technical Field

[0001] This invention belongs to the technical field of metal-organic framework materials as drug delivery carriers, specifically relating to a GSH / pH dual-responsive Cu2O@Cu 2+ / ZIF-67@ZnPc core-shell structured nanomedicines, their preparation methods, and applications. Background Technology

[0002] ROS, as a byproduct of oxygen metabolism, plays a role in cell signaling and maintaining cellular oxidative homeostasis. Currently, regulating ROS levels to induce cell death has been proven to be an effective cancer treatment method, and many ROS-based cancer therapies have been extensively studied.

[0003] Photodynamic therapy (PDT) is a clinically approved minimally invasive treatment for reactive oxygen species (ROS). It utilizes light of a specific wavelength to irradiate photosensitizers (PS) to generate ROS, thereby exerting selective cytotoxicity on malignant cells. Commonly used PSs, as key components of PDT, typically exhibit hydrophobicity and a tendency to aggregate. For example, phthalocyanines (Pc) are planar fused-ring compounds with high singlet oxygen production rates, which readily aggregate in physiological environments, leading to fluorescence quenching (ACQ) and hindering ROS generation. Reducing the aggregation of such photosensitizers can improve the efficiency of PDT. Metal-organic frameworks (MOFs) are porous nanomaterials with high specific surface area, tunable pore size, and excellent biocompatibility. Furthermore, the pores or defects in MOFs can be used to "immobilize" PSs to prevent their aggregation. Therefore, using MOFs as carriers for such PSs can effectively reduce aggregation and improve the efficacy of PDT.

[0004] The tumor microenvironment (TME) is characterized by hypoxia, acidic pH, glutathione (GSH) overexpression, and high H2O2 levels. Low endogenous O2 levels limit the photodynamic therapy effect of photosensitizers in tumor cells, and GSH, as an intracellular free radical scavenger, further depletes PDT-generated glutathione (PDT). 1 O2 reduces photodynamic activity, making PDT (photodynamic therapy) alone insufficient to achieve satisfactory therapeutic effects. Chemokinetic therapy (CDT) utilizes Fenton or Fenton-like reactions to convert excess hydrogen peroxide (H2O2) within cancer cells into highly cytotoxic hydroxyl radicals (·OH), thereby killing cancer cells. More importantly, with the generation of ·OH, some Fenton or Fenton-like reactions can induce O2 production, alleviating TME (tumor emphysema) hypoxia and further enhancing the therapeutic effect of PDT, achieving synergistic combined therapy with CDT.

[0005] Traditional Fenton metal Fe 2+Fenton activity at pH 2-4, while Cu + has a wider pH application range and higher Fenton-like catalytic activity, with a reaction rate about 160 times higher than the former. However, the clinical toxicity of Cu + limits its application in tumor therapy. Cu2O as a Cu + ion source has high clinical toxicity. If a TME-responsive shell is formed on the surface of Cu + source to prevent Cu + from leaking in advance, it will help to selectively enrich in tumor sites, improve clinical toxicity, and fully exert Fenton-like catalytic activity. Some functional MOFs not only can load drugs through their porous structure, but also can be specifically degraded in TME, so functional MOFs as a coating shell can not only protect the core from being attacked by the immune system, but also as a stable controlled release framework to accurately release the contents at the tumor site. However, current research on TME-responsive MOF shells still mainly focuses on ZIF-8, MIL-100(Fe) and other structures that are easy to modify and modify, and the development of other more functional MOFs is very limited. Due to the large interface energy barrier between the core and the shell materials, MOFs are more likely to phase separately in solution or the core is scattered on the surface of MOFs when synthesizing MOF shell structure on the surface of Cu2O core. At the same time, there are problems such as early leakage of Cu + / drugs, uneven distribution of shell and core, poor repeatability and stability, etc. Therefore, it is still challenging to develop functional MOF composite materials with uniform core-shell structure and TME responsiveness.

[0006] ZIF-67 is a MOF formed by Co 2+ and 2-methylimidazole, which has good ability to accommodate guest drug molecules and biocompatibility, and can be degraded in acidic tumor microenvironment to release its contents. High-valent Fenton metal ions (such as Fe 3+ , Cu 2+ , Mn 4+ ) can consume GSH and be reduced to low valence state, and produce ·OH by further reaction with H2O2 for CDT therapy. At the same time, the consumption of antioxidant GSH is beneficial to enhance the level of ROS in tumor cells, thereby synergistically improving the PDT treatment effect. Therefore, it is of great practical significance to coat ZIF-67 structure doped with high-valent Fenton ions on the surface of Cu2O to construct a GSH / pH dual-responsive nanocarrier.

[0007] Cu2O as a Cu +There are few reports on the combination of ion source and MOFs for CDT / PDT synergistic therapy. If ZIF-67 is used to form a core-shell drug carrier, the clinical toxicity can be improved, and the loading efficiency of photosensitizer can be improved. More importantly, in the present application, Cu 2+ The doped core-shell MOFs structure protects the Fenton reagent Cu2O and efficiently loads the photosensitizer drug, has GSH / pH response ability, can increase the ROS level in tumor cells by consuming antioxidant GSH, and finally releases the contents for efficient CDT / PDT synergistic therapy. SUMMARY

[0008] The purpose of the present application is to provide a GSH / pH dual-responsive Cu2O@Cu 2+ / ZIF-67@ZnPc core-shell structure nanodrug and a preparation method and application thereof. In the TME, the GSH / pH response releases the Fenton reagent and the photosensitizer, and reduces the antioxidant GSH in the TME, so as to achieve efficient CDT / PDT synergistic therapy.

[0009] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0010] A Cu2O@Cu 2+ / ZIF-67@ZnPc core-shell structure nanodrug synthesized based on a Cu2O self-template method, and a synthesis method thereof, the synthesis method comprising the following steps:

[0011] (1) A certain amount of copper chloride dihydrate and polyvinylpyrrolidone K30 are put into a flask, 50 mL of deionized water is added, and stirring is carried out at room temperature for 15 min until the solution is clear. 5 mL of sodium hydroxide aqueous solution is added to the solution, and stirring is carried out at room temperature for 5 min. Then, 5 mL of ascorbic acid aqueous solution is added, and stirring is carried out at room temperature for 15 min to obtain Cu2O. After centrifugation and washing with water and methanol for several times, Cu2O is dispersed in 15 mL of methanol.

[0012] (2) The solution of step (1) is mixed with sodium dodecylsulfate and ultrasonicated for 30 min, then centrifuged and dispersed in 37 mL of methanol. 12 mL of cobalt nitrate hexahydrate methanol solution is added, and stirring is carried out at room temperature for 10 min. Then, 12 mL of 2-methylimidazole methanol solution is added, and stirring is carried out at room temperature for 1 h. After centrifugation and washing with methanol, Cu2O@Cu 2+ / ZIF-67 nanomaterial is obtained by drying in a vacuum drying box.

[0013] (3) A part of the product Cu2O@Cu 2+ / ZIF-67 was dissolved in 5 mL of methanol, and zinc tetracarboxyphthalocyanine was dissolved in 2 mL of N,N-dimethylformamide. The two solutions were mixed and stirred at room temperature, centrifuged, and washed with N,N-dimethylformamide and methanol before being dried in a vacuum drying oven.

[0014] (4) Dissolve the product of step (3) in 10 mL of deionized water and mix it with the addition polymer of polypropylene glycol and ethylene oxide. Stir at room temperature for 12 h, centrifuge and dry in a vacuum drying oven to obtain the core-shell structured nanomedicine.

[0015] Preferably, in step (1), the polyvinylpyrrolidone K30 added is 1.7%~2.3% of the solution mass, and the molar ratio of copper chloride dihydrate: sodium hydroxide: ascorbic acid is 2:10:5. In step (2), after adding sodium dodecyl sulfonate, its mass concentration is 0.1 mg / mL, and the molar ratio of cobalt nitrate hexahydrate: 2-methylimidazole is 3:25. In step (3), Cu2O@Cu 2+ The mass concentration of / ZIF-67 is 2~2.5 mg / mL, which corresponds to a mass concentration of 2~2.5 mg / mL of zinc tetracarboxylated phthalocyanine. The mass concentration of the polypropylene glycol and ethylene oxide addition polymer added in step (4) is 1.8~2.5 mg / mL.

[0016] GSH / pH dual-responsive Cu2O@Cu prepared by the method described above 2+ Application of ZIF-67@ZnPc core-shell structured nanomedicines in the preparation of photodynamic therapy nanomedicines.

[0017] This invention utilizes the fact that Cu2O will release Cu under certain conditions. + And it is oxidized to Cu 2+ Using Cu₂O nanospheres as templates, sodium dodecyl sulfonate was used to change the potential, and Co was adsorbed through electrostatic adsorption. 2+ After adsorbing onto the Cu2O surface, the 2-methylimidazolium ligand reacts with Co. 2+ and Cu released from Cu2O + Further reaction forms Cu 2+ The structure of doped ZIF-67 MOFs. Due to Cu 2+ doping, Cu 2+ / ZIF-67 can deplete the high levels of GSH in tumor cells, thereby promoting the effect of PDT by weakening the antioxidant activity in the TME. Meanwhile, Cu2O and Co... 2+ It can generate ·OH and O2 through Fenton-like reactions and Haber-Weiss reactions, thereby working together with the photosensitizer ZnPc released in MOFs to achieve the purpose of CDT / PDT synergistic therapy.

[0018] The beneficial effects of this invention are as follows:

[0019] (1) Synthesizing Cu by adding surfactants to the surface of Cu2O 2+ The doped ZIF-67 provides a new and universal method for synthesizing doped MOF structures based on the metal oxide self-templation method.

[0020] (2) Synthesized GSH / pH dual-response Cu 2+ The ZIF-67 shell can consume the antioxidant GSH in the TME, improving the therapeutic effect of ROS and addressing the issue of phthalocyanine photosensitizers' tendency to aggregate. The core-shell structure of functional MOFs can increase drug loading while reducing the off-target toxicity of nanomedicines.

[0021] (3) Using Cu + The advantage of being able to react and generate ·OH and O2 in the tumor microenvironment, combined with the photosensitizer ZnPc released from MOFs, forms a CDT / PDT synergistic therapy. Attached Figure Description

[0022] Figure 1 Cu2O@Cu in Example 1 2+ XRD pattern of ZIF-67 core-shell nanomaterials.

[0023] Figure 2 Cu2O@Cu in Example 1 2+ (a) SEM image of the ZIF-67 core-shell nanomaterial, (b) TEM image, and (c) SEM image of the material without sodium dodecyl sulfonate in the reaction.

[0024] Figure 3 Different concentrations of Cu2O@Cu were used in Example 1. 2+ / ZIF-67 core-shell structured nanomaterials' ability to consume GSH test graph.

[0025] Figure 4 Cu2O@Cu in Example 1 2+ (a) ·OH generation capacity of ZIF-67 core-shell nanomaterial in the presence of H2O2, (b) O2 generation capacity in the presence of H2O2.

[0026] Figure 5 Cu2O@Cu in Example 1 2+ A graph illustrating the singlet oxygen generation capacity of / ZIF-67@ZnPc core-shell nanomedicines under acidic conditions.

[0027] Figure 6 Cu2O@Cu in Example 1 2+ The dark toxicity (a) and phototoxicity (b) of the ZIF-67@ZnPc core-shell nanomedicine on HepG2 cells.

[0028] Figure 7 Cu2O@Cu 2+ The dark toxicity of the ZIF-67@ZnPc core-shell structure nanodrug on LO2 cells and HepG2 cells. DETAILED DESCRIPTION

[0029] In order to make the content of the present application more convenient to understand, the technical solutions of the present application will be further described below in combination with specific embodiments, but the present application is not limited thereto.

[0030] Example 1

[0031] (1) 0.17 g of copper chloride dihydrate and 1 g of polyvinylpyrrolidone K30 were taken into a flask, 50 mL of ionized water was added, and stirring was carried out at room temperature for 15 min until the solution was clear. 5 mL of a 2M sodium hydroxide aqueous solution was added to the solution, and stirring was carried out at room temperature for 5 min. Subsequently, 5 mL of a 1M ascorbic acid aqueous solution was added, and stirring was carried out at room temperature for 15 min to obtain Cu2O. After centrifugation at 13000 rpm for 5 min and washing twice with water and methanol, Cu2O was dispersed in 15 mL of methanol;

[0032] (2) The solution of step (1) was mixed with 0.15 g of sodium dodecyl sulfonate and ultrasonically treated for 30 min, and then centrifuged. The obtained product was dispersed in 37 mL of methanol, 12 mL of a cobalt nitrate hexahydrate methanol solution (0.008 g / mL) was added, and stirring was carried out at room temperature for 10 min. Subsequently, 12 mL of a 2-methylimidazole methanol solution (0.017 g / mL) was added, and stirring was carried out at room temperature for 1 h. After centrifugation at 10000 rpm for 5 min and washing twice with methanol, Cu2O@Cu 2+ / ZIF-67 was obtained.

[0033] (3) 10 mg of Cu2O@Cu 2+ / ZIF-67 was dissolved in 5 mL of methanol, 4 mg of zinc tetracarboxy phthalocyanine was dissolved in 2 mL of N,N-dimethylformamide, and the two solutions were mixed and stirred at room temperature. After centrifugation at 10000 rpm for 5 min and washing once with N,N-dimethylformamide and twice with methanol, the obtained product was dried in a vacuum drying box.

[0034] (4) The product of step (3) was dissolved in 10 mL of ionized water and mixed with 20 mg of a polypropylene glycol and ethylene oxide polyadduct. Stirring was carried out at room temperature for 12 h. After centrifugation at 10000 rpm for 5 min, the obtained product was dried in a vacuum drying box to obtain the core-shell structure nanodrug.

[0035] Example 2

[0036] (1) Take 0.17 g of copper chloride dihydrate and 0.9 g of polyvinylpyrrolidone K30 into a flask, add 50 mL of ionized water, stir at room temperature for 15 min until the solution is clear, add 5 mL of 2M sodium hydroxide aqueous solution to the solution, stir at room temperature for 5 min, then add 5 mL of 1M ascorbic acid aqueous solution, stir at room temperature for 15 min, obtain Cu2O, centrifuge at 13000 rpm for 5 min and wash twice with water and methanol, then disperse in 15 mL of methanol;

[0037] (2) Mix the solution of step (1) with 0.15 g of sodium dodecyl sulfate and ultrasonic for 30 min, then centrifuge, disperse in 37 mL of methanol, add 12 mL of cobalt nitrate hexahydrate methanol solution (0.008 g / mL), stir at room temperature for 10 min, then add 12 mL of 2-methylimidazole methanol solution (0.017 g / mL), stir at room temperature for 1 h, centrifuge at 10000 rpm for 5 min and wash twice with methanol, then dry in a vacuum drying oven to obtain Cu2O@Cu 2+ / ZIF-67 nanomaterials;

[0038] (3) Take 12 mg of Cu2O@Cu 2+ / ZIF-67 dissolved in 5 mL of methanol, take 5 mg of zinc tetracarboxy phthalocyanine dissolved in 2 mL of N,N-dimethylformamide, mix the two solutions and stir at room temperature, centrifuge at 10000 rpm for 5 min and wash once with N,N-dimethylformamide and twice with methanol, then dry in a vacuum drying oven;

[0039] (4) Dissolve the product of step (3) in 10 mL of ionized water and mix with 23 mg of polypropylene glycol and ethylene oxide adduct, stir at room temperature for 12 h, centrifuge at 10000 rpm for 5 min and dry in a vacuum drying oven to obtain the core-shell structure nanodrug.

[0040] Figure 1 The XRD pattern of the Cu2O@Cu 2+ / ZIF-67 core-shell structure nanomaterials in Example 1, it can be seen that the diffraction peaks of the synthesized core-shell structure nanomaterials correspond to the peaks of Cu2O and ZIF-67. Figure 1

[0041] Figure 2 The SEM and TEM images of the Cu2O@Cu 2+ / ZIF-67 core-shell structure nanomaterials in Example 1, if sodium dodecyl sulfate is not added in the reaction, two phases are formed. It may be that when the potential of Cu2O is not changed by using sodium dodecyl sulfate, the Co 2+ ​Ions are more inclined to be uniformly dispersed in the solution, resulting in the subsequent 2-methyl imidazole ligand and its single phase in solution. This illustrates the necessity of this synthesis method.

[0042] Figure 3 GSH consumption ability of Cu2O@Cu 2+ / ZIF-67 core-shell structure nanomaterials was tested using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to detect the GSH consumption ability of MOF. DTNB can react with GSH to generate 5-mercapto-2-nitrobenzoic acid (TNB), which has a maximum absorption at 412 nm. Therefore, we can determine the degree of GSH consumption by measuring the decrease in absorption at 412 nm in the reaction solution. As can be seen in the figure, the absorbance at 412 nm gradually decreases with increasing material concentration, which proves that the doping of Cu 2+ can effectively consume GSH.

[0043] Figure 4 GSH consumption ability of Cu2O@Cu 2+ / ZIF-67 core-shell structure nanomaterials (a) ·OH generation ability in the presence of H2O2, (b) O2 generation ability in the presence of H2O2. Methylene blue (MB) was used as a ·OH probe to observe the decrease in its absorption peak at 664 nm to detect the generation of ·OH. By observing the decrease in the absorption peak of MB at 664 nm, it can be seen that the nanomaterial has excellent ·OH generation ability for CDT treatment. The dissolved oxygen concentration in the aqueous solution in the presence of H2O2 was tested using a portable dissolved oxygen meter, and it can be seen that the material can generate O2 to enhance the PDT effect.

[0044] Figure 5 GSH consumption ability of Cu2O@Cu 2+ / ZIF-67@ZnPc core-shell structure nanomedicine singlet oxygen generation ability in acidic environment. 1,3-diphenyl isobenzofuran (DPBF) was used to test the singlet oxygen generation ability of nanomedicine under 670 nm laser irradiation. DPBF can react with O2 to cause a decrease in its absorption peak at 420 nm. By observing the decrease in absorbance at 420 nm, it can be seen that the nanomedicine can release the photosensitizer ZnPc in the acidic environment to perform PDT treatment. 1

[0045] Figure 6 GSH consumption ability of Cu2O@Cu 2+ / ZIF-67@ZnPc core-shell structure nanomedicine MTT method cell phototoxicity and dark toxicity experiment on HepG2 cells. From Figure 6 a, it can be seen that Cu2O@Cu 2+ ​ / ZIF-67 has CDT effect, and the CDT / PDT combined treatment formed after loading photosensitizer ZnPc has obvious synergistic effect compared with single treatment mode.

[0046] Figure 7 Cu2O@Cu 2+ The MTT method cell dark toxicity experiment of the / ZIF-67@ZnPc core-shell structure nano drug on LO2 cells shows that the material has good biocompatibility.

[0047] The above only describes the preferred embodiments of the present application, and any equivalent changes and modifications made within the scope of the present application should be included in the scope of the present application.

Claims

1. A GSH / pH dual-responsive Cu2O@Cu 2+ The application discloses a preparation method of a GSH / pH dual-responsive Cu2O@Cu The Cu2O@Cu / ZIF-67 core-shell structure is prepared by mixing self-made Cu2O nanospheres as a self-template, a cobalt nitrate hexahydrate solution and a 2-methyl imidazole methanol solution 2+ The Cu2O@Cu / ZIF-67 core-shell structure is prepared by mixing self-made Cu2O nanospheres as a self-template, a cobalt nitrate hexahydrate solution and a 2-methyl imidazole methanol solution 2+ The Cu2O@Cu / ZIF-67 core-shell structure is prepared by mixing self-made Cu2O nanospheres as a self-template, a cobalt nitrate hexahydrate solution and a 2-methyl imidazole methanol solution The method comprises the following steps: (1) dissolving copper chloride dihydrate and polyvinylpyrrolidone K30 in 50 mL of deionized water, stirring at room temperature until the solution is clear, adding 5 mL of sodium hydroxide solution and 5 mL of ascorbic acid solution, stirring at room temperature for 15 min, obtaining Cu2O nanospheres, centrifuging and dispersing in 15 mL of methanol after washing with water and methanol for several times; (2) The solution obtained in step (1) is mixed with sodium dodecyl sulfate, ultrasonic treatment for 30 min, then centrifuged, dispersed in 37 mL of methanol, 12 mL of cobalt nitrate hexahydrate methanol solution is added, stirred at room temperature for 10 min, then 12 mL of 2-methylimidazole methanol solution is added, stirred at room temperature for 1 h, centrifuged and washed with methanol, and then vacuum dried to obtain Cu2O@Cu 2+ / ZIF-67 nanomaterials; (3) Cu2O@Cu 2+ / ZIF-67 nanomaterials were dissolved in 5 mL of methanol to obtain solution A, and zinc tetracarboxy phthalocyanine was dissolved in 2 mL of N,N-dimethylformamide to obtain solution B. Solution A and B were mixed, stirred at room temperature, centrifuged, washed with N,N-dimethylformamide and methanol, and then vacuum dried. (4) The product obtained in step (3) was dissolved in 10 mL of deionized water and mixed with a polyadduct of polypropylene glycol and ethylene oxide, stirred at room temperature for 12 h, centrifuged and vacuum dried to obtain the Cu2O@Cu 2+ ZIF-67@ZnPc core-shell structure nanodrug; The mass concentration of sodium dodecyl sulfonate in step (2) is 0.1 mg / mL, and the molar ratio of cobalt nitrate hexahydrate to 2-methylimidazole is 3:

25.

2. The method of claim 1, wherein: The amount of polyvinylpyrrolidone K30 in step (1) is 1.7% to 2.3% of the mass of the solution, and the molar ratio of copper chloride dihydrate to sodium hydroxide to ascorbic acid is 2:10:

5.

3. The method of claim 1, wherein: Cu2O@Cu 2+ The mass concentration of ZIF-67 was 2-2.5 mg / mL, and the mass concentration of zinc tetracarbonyl phthalocyanine was 2-2.5 mg / mL.

4. The method of claim 1, wherein: The mass concentration of the polyadduct of polypropylene glycol and ethylene oxide in step (4) is 1.8-2.5 mg / mL.

5. The GSH / pH dual-responsive Cu20@Cu / ZIF-67@ZnPc core-shell structure nanodrug prepared by the method of claim 1. 2+ The GSH / pH dual-responsive Cu20@Cu / ZIF-67@ZnPc core-shell structure nanodrug is characterized in that: The nanodrug is a dodecahedron with a size of 200-300 nm and a thickness of 50-100 nm.

6. The GSH / pH dual-responsive Cu2O@Cu2O@ZIF-67@ZnPc core-shell structure nanodrug prepared by the method of claim 1. 2+ The application of the GSH / pH dual-responsive Cu2O@Cu2O@ZIF-67@ZnPc core-shell structure nanodrug in the preparation of a photodynamic therapy nanodrug.

Images