Preparation method and application of nanomedicine for multi-modal synergistic tumor treatment

Abstract

The present invention discloses a method for preparing and applying a nanomedicine for multimodal synergistic tumor therapy, relating to the field of biomedical materials technology. The key technical features include: Step S1: preparing a biligand metal-organic framework (MOF) using a hydrothermal method; Step S2: dispersing the MOF in 10 mL of anhydrous ethanol, adding manganese(I) bromide pentacarbonyl, magnetically stirring, and thoroughly washing with anhydrous methanol to remove the unloaded Mn(I) bromide pentacarbonyl; finally, obtaining a MOF@Mn(I) bromide pentacarbonyl; Step S3: ultrasonically dissolving CuCl2·2H2O and sodium citrate dihydrate in 50 mL of aqueous solution; and Step S4: preparing a MOF@Mn(I) bromide pentacarbonyl@copper sulfide. The proposed nanomedicine has a stable structure and can be precisely released in complex tumor microenvironments, enabling convenient multimodal synergistic tumor therapy, achieving excellent therapeutic effects while reducing the side effects of each single treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a preparation method and application of a nanomedicine for multi-modal synergistic tumor treatment. Background Art

[0002] With the continuous advancement of cancer treatment technologies, the limitations of single treatment modalities are becoming increasingly apparent. Traditional chemotherapy and radiotherapy, while killing tumor cells, often inevitably damage normal tissues, leading to serious side effects. Furthermore, due to the heterogeneity and complex microenvironment of tumors, single treatments are often unable to completely eliminate tumors, which can easily lead to drug resistance and recurrence. To overcome these challenges, multimodal synergistic treatment strategies have gradually become a research hotspot in cancer treatment.

[0003] Nanomedicines, due to their unique physicochemical properties, such as high surface area, excellent biocompatibility, and tunable drug release, are ideal vehicles for multimodal synergistic therapies. Through targeted design, nanomedicines can integrate multiple treatment modalities, such as chemotherapy, photothermal therapy, photodynamic therapy, and immunotherapy, onto a single nanoplatform, enhancing synergistic therapeutic effects. Furthermore, their drug-loading capacity and controlled release mechanisms can help minimize damage to normal cells during treatment, improving patients' quality of life.

[0004] However, how to efficiently prepare multifunctional nanomedicines and achieve their precise release and multimodal synergistic effects in the complex tumor microenvironment remains a pressing technical challenge. This paper proposes an innovative nanomedicine preparation method designed to optimize the structure and function of nanocarriers, enabling multimodal synergistic tumor therapy and thus enhancing the overall effectiveness of tumor treatment. Summary of the Invention

[0005] The purpose of the present invention is to solve the above problems and provide a method for preparing and applying nanomedicine for multi-modal synergistic tumor treatment.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The present invention provides a method for preparing a nanomedicine for multi-modal synergistic tumor treatment, comprising the following steps:

[0008] Step S1, preparing a biligand metal-organic framework by a hydrothermal method: tetrakis(4-carboxyphenyl)porphine ferric chloride, 2,2'-bipyridine-5,5'-dicarboxylic acid, ZrOCl2·8H2O, and benzoic acid were ultrasonically dissolved in a round-bottom flask containing N,N-dimethylformamide; the reaction solution was stirred at 90°C for 3 hours; the oil bath was covered with aluminum foil to avoid light exposure; after the reaction was completed, the nanoparticles were collected by centrifugation and washed three times with fresh N,N-dimethylformamide and anhydrous ethanol respectively; finally, the product was stored in the dark for further use;

[0009] Step S2: Dispersing the biligand metal-organic framework in 10 mL of anhydrous ethanol, adding pentacarbonyl manganese (I) bromide, and magnetic stirring; after uniformly mixing at room temperature for 12 hours, the mixture is heated to 75° C. and maintained at this temperature for another 4 hours; collecting the orange product by centrifugation and thoroughly washing with anhydrous methanol to remove the unloaded pentacarbonyl manganese (I) bromide; finally, placing the product biligand metal-organic framework @ pentacarbonyl manganese (I) bromide in the dark for further use;

[0010] Step S3: Dissolve CuCl2·2H2O and sodium citrate dihydrate in 50 ml of aqueous solution under ultrasonication; then, add 50 μL of sodium sulfide solution at room temperature with stirring for 5 minutes; then, heat the reaction solution at 90°C for 30 minutes until a dark green solution is obtained; transfer the mixture into ice water; obtain CuS and store at 4°C;

[0011] Step S4: For the biligand metal-organic framework @ pentacarbonyl manganese (I) bromide @ copper sulfide, the prepared CuS aqueous solution is mixed with the biligand metal-organic framework @ pentacarbonyl manganese (I) bromide and stirred for 1 hour, then transferred to 90°C in an oil bath and stirred for 4 hours; the product biligand metal-organic framework @ pentacarbonyl manganese (I) bromide @ copper sulfide is collected by centrifugation and washed with deionized water; 60 μL of 2 mg / mL biligand metal-organic framework @ pentacarbonyl manganese (I) bromide @ copper sulfide is resuspended in 2 mM PBS to obtain the drug.

[0012] The present invention is further configured as follows: in step S1, N,N-dimethylformamide is 1-5 ml, tetrakis(4-carboxyphenyl)porphine ferric chloride is 0.1-1 mg, 2,2'-bipyridine-5,5'-dicarboxylic acid is 20-40 mg, ZrOCl2·8H2O is 10-30 mg, and benzoic acid is 50-300 mg.

[0013] The present invention is further configured as follows: in step S2, the amount of the biligand metal organic framework is 10-40 mg, and the amount of manganese(I) bromide pentacarbonyl is 10-50 mg.

[0014] The present invention is further configured as follows: in step S3, the amount of CuCl2·2H2O is 5-20 mg, the amount of sodium citrate dihydrate is 5-20 mg, and the amount of sodium sulfide solution is 100-300 mg / mL.

[0015] The present invention is further configured as follows: in step S4, the CuS aqueous solution is 3-10 ml, and the biligand metal organic framework@pentacarbonyl manganese(I) bromide is 1-5 mg.

[0016] A nanomedicine for multi-modal synergistic tumor treatment, the nanomedicine comprising 2,2'-bipyridine-5,5'-dicarboxylic acid, tetrakis(4-carboxyphenyl)porphine ferric chloride, pentacarbonyl manganese bromide and copper sulfide

[0017] Application of a nanomedicine in multimodal synergistic tumor therapy.

[0018] Compared with the existing technology, the beneficial effects of this solution are: the nanomedicine proposed in the present invention has a stable structure, can be accurately released in the complex tumor microenvironment, can conveniently carry out multi-modal synergistic treatment of tumors, achieve good therapeutic effects while reducing the side effects of each single treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the preparation and use process in an embodiment of the present invention;

[0020] Figure 2 This is a demonstration of the feasibility of multiple therapeutic applications of nanomedicines in solution according to the present invention. (A) is a schematic diagram of the in vitro multiple therapeutic effect evaluation based on DMCH, (B) is the fluorescence response of DMCH to the singlet oxygen green fluorescent probe (Singlet Oxygen Sensor Green, SOSG) at different times under 660nm laser irradiation or without irradiation, and (C) is the probe system (5μM CO probe + 20μM PdCl2 + 500μg / mL Fluorescence spectra of DMCH in PBS buffer (10 mM, pH 7.4, 1% DMSO, v / v) at 37°C with or without the addition of H2O2 (1 mM). (D) UV-Vis absorption spectra of aqueous 3,3',5,5'-tetramethylbenzidine (TMB) solutions after different treatments. (E) UV-Vis absorption of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) for determining GSH levels. (F) Thermal images of PBS or DMCH solutions at different times and laser power densities. (G) Laser power density-dependent photothermal properties of DMCH (500 μg / mL). (H) 660 nm laser (0.86 W / cm 2) thermal cycling curves under irradiation, (I) is the photothermal and cooling curves of DMCH suspension (500 μg / mL), (J) is the curve of cooling time versus the negative natural logarithm of temperature driving force, all data demonstrate its potential for multiple combination therapies in advance;

[0021] Figure 3 The therapeutic effect of nanomedicine in nude mouse tumors in the embodiment of the present invention is shown in Figure 2, where (A) is a schematic diagram of the treatment of tumor-bearing mice, and (B) is a diagram of tumor-bearing mice treated with PBS and PMCH and then irradiated with 808 nm laser (1 W / cm 2 ) Infrared thermal imaging at different times after irradiation, (C) imaging examination of five organs and tumors 24 hours after treatment, (D) changes in mouse body weight within 13 days of treatment, (E) time-dependent tumor growth curve of each treatment group, (F) average tumor weight of each group, (G) photos of tumor tissues of mice in different groups after different treatments: (I) PBS+660nm+808nm irradiation group, (II) DMCH group, (III) DMCH+660nm group, (IV) DMCH+808nm group, (V) DMCH+660nm+808nm group. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0023] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0024] Example:

[0025] (1) DMOF (biligand metal-organic framework) was prepared by hydrothermal method. 0.53 mg of tetrakis(4-carboxyphenyl)porphine ferric chloride (TCPP-Fe(Ⅲ)), 31.7 mg of 2,2'-bipyridine-5,5'-dicarboxylic acid (BPyDC), 20 mg of ZrOCl2·8H2O, and 200 mg of benzoic acid (BA) were dissolved by ultrasonication in a round-bottom flask containing 3 ml of N,N-dimethylformamide (DMF). The reaction solution was stirred at 90°C for 3 h. The oil bath was covered with aluminum foil to avoid light exposure. After the reaction was completed, the nanoparticles were collected by centrifugation and washed three times with fresh DMF and anhydrous ethanol respectively. Finally, the product was stored in the dark for further use.

[0026] (2) 20 mg of DMOF was dispersed in 10 mL of anhydrous ethanol, and 30 mg of pentacarbonyl manganese(I) bromide (MnCO) was added and magnetically stirred. After uniform mixing at room temperature for 12 h, the mixture was heated to 75 °C and maintained at this temperature for another 4 h. The orange product was collected by centrifugation and thoroughly washed with anhydrous methanol to remove the unloaded MnCO. Finally, the product, biligand metal-organic framework@pentacarbonyl manganese(I) bromide (DMOF@MnCO,DM), was placed in the dark for further use.

[0027] (3) 10.8 mg of CuCl2·2H2O and 11.4 mg of sodium citrate dihydrate were dissolved in 50 mL of aqueous solution by ultrasound. Subsequently, 50 μL of sodium sulfide solution (242 mg / mL) was added with stirring at room temperature for 5 min. The reaction solution was then heated at 90°C for 30 min until a dark green solution was obtained. The mixture was transferred to ice water. CuS was obtained and stored at 4°C.

[0028] (4) For DMOF@MnCO@CuS, a 100 μg / mL aqueous solution of CuS (5 mL) was mixed with DMOF@MnCO (1 mg) and stirred for 1 h. The mixture was then transferred to 90°C in an oil bath and stirred for 4 h. The resulting DMC was collected by centrifugation and washed with deionized water. DMC was resuspended in 2 mM PBS (2 mg / mL, 60 μL) and injected into the tail vein of mice for the corresponding intervention and treatment.

[0029] In order to evaluate the photodynamic therapy (PDT), photothermal therapy (PTT), chemodynamic therapy (CDT) and gas therapy (GAT) capabilities of DMCH, we designed in vitro assays, such as Figure 2 As shown in A. First, the singlet oxygen sensor green fluorescent probe (SOSG) was used to evaluate the generation of singlet oxygen ( 1 O2) capability, the probe is 1 After oxidation by O2, it emits 525nm fluorescence. Figure 2 As can be seen from the blue curve in B, in a solution containing 0.5 mg / mL DMCH, the fluorescence signal of 25 μM SOSG did not increase significantly without 660 nm laser irradiation. However, under 660 nm laser irradiation, the fluorescence intensity of SOSG increased rapidly and reached a plateau within 3 minutes ( Figure 2 B red curve), which is 3.5 times higher than that of the group without 660 nm laser irradiation. This shows that DMCH has a high efficiency in PDT. 1 O2 capability. In addition, MnCO as a CO release agent can trigger the release of CO gas molecules in the presence of H2O2. Therefore, we use the CO probe to detect the released CO. Figure 3As shown in Figure C, in a solution containing 5 μM CO probe and 1 mM H2O2, the fluorescence was enhanced by about 3.9 times, indicating that H2O2 can trigger the release of CO from DMCH. The generation of highly toxic ·OH radicals was measured using a standard colorimetric method based on TMB, demonstrating the activation of DMCH as a Fenton reagent for CDT. The generated ·OH can catalyze the oxidation of TMB to generate oxidized TMB (oxTMB), which exhibits a characteristic absorbance at 650 nm. Figure 2 As shown in D, in the absence of 0.5 mg / mL DMCH (red) or 1 mM H2O2 (black), the TMB solution remains inactive. In the presence of 0.5 mg / mL DMCH and 1 mM H2O2, the TMB solution appears blue, showing the characteristic absorption curve of oxTMB, with the highest absorbance value at 652 nm. After further addition of 0.01 mM GSH, the absorbance increased (green), indicating that the addition of GSH to the solution can promote the metal ion reduction reaction and enhance the effectiveness of CDT. This enhancement cannot be observed in the absence of DMCH (pink). We further used the thiol (-SH) indicator DTNB to measure the consumption of glutathione (GSH). From Figure 2 E can be seen that when DMCH was added alone, the absorbance curve decreased slightly (black) compared with the solution with 10mM GSH added alone (red), indicating that inactive DMCH could not significantly consume GSH. When H2O2 and GSH were added to 0.5mg / mL DMCH, GSH was rapidly reduced by DMCH, and the color of the solution changed from yellow to colorless. This suggests that DMCH can effectively reduce the level of intracellular GSH and improve the therapeutic effect of CDT. In order to evaluate the photothermal conversion ability of DMCH in aqueous solution, 808nm laser was used at different power densities (0.30, 0.60, 0.86 and 1W / cm 2 ) irradiated DMCH, and deionized water was used as blank control ( Figure 2 F). The temperature rise is positively correlated with the irradiation power and irradiation time ( Figure 2 F and G). At 0.86W / cm 2 The photothermal stability of DMCH was confirmed by the repeated observation of peak temperature during 5 heating / cooling cycles at a power density of 100 nm. Figure 2 H), indicating that DMCH maintained strong photothermal stability during the entire experiment. 2 The aqueous dispersion of DMCH was irradiated with a near-infrared laser until it reached a steady-state temperature and then cooled to room temperature after the laser was turned off ( Figure 2 I). By the cooling curve ( Figure 2 J) According to the equation in the experimental section, the photothermal conversion efficiency (η) of DMCH is estimated to be 39.9%.

[0030] Based on the synergistic therapeutic effects and favorable biodistribution of DMCH observed in vitro, we extended our evaluation to assess its antitumor efficacy in vivo. Figure 3 A. Tumor-bearing mice were treated with DMCH via tail vein injection. 24 hours after injection, the tumor site was exposed to 808nm near-infrared laser light for 10 minutes, and thermal imaging was used to capture temperature changes. The surface temperature of the tumor after DMCH treatment increased rapidly within 10 minutes, reaching a core temperature of 37.9°C, significantly exceeding the 46.6°C after PBS treatment ( Figure 3 B). We studied the biodistribution of DMCH by intravenously injecting only Cy5-modified hairpin probes, and then removed tumors and major organs for ex vivo fluorescence imaging. Cy5 fluorescence was the most intense in all tumor-bearing organs except the liver ( Figure 3 C), indicating that DMCH is efficiently accumulated in the tumor. The liver metabolizes DMCH efficiently, which indicates reduced long-term toxicity to the organism. Body weight and tumor volume were measured daily during treatment. No significant changes in body weight were observed in any treatment group ( Figure 3 D), indicating that the side effects of the nanoplatform are negligible. As can be seen from the tumor volume curve, the group receiving combined treatment (Group V) has the most significant inhibition of tumor growth compared with the control group and other single treatment groups ( Figure 3 E). After treatment, tumor tissues of each group were collected. Tumor weight ( Figure 3 F) and representative photos ( Figure 3 G) confirmed the conclusion that the combined treatment group had a better inhibitory effect on tumor growth.

[0031] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the embodiments as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A method for preparing nanomedicine for multi-modal synergistic tumor treatment, characterized in that: The following steps are involved: Step S1: Preparation of a biligand metal-organic framework using a hydrothermal method: Tetrakis(4-carboxyphenyl)porphine ferric chloride, 2,2'-bipyridine-5,5'-dicarboxylic acid, ZrOCl2·8H2O, and benzoic acid were ultrasonically dissolved in a round-bottom flask containing N,N-dimethylformamide; the reaction solution was stirred at 90°C for 3 h; the oil bath was covered with aluminum foil to avoid light exposure; after the reaction was completed, the nanoparticles were collected by centrifugation and washed three times with fresh N,N-dimethylformamide and anhydrous ethanol respectively; finally, the product was stored in the dark for further use; Step S2: Disperse the biligand metal-organic framework in 10 mL of anhydrous ethanol, add pentacarbonyl manganese bromide, and stir magnetically; after uniformly mixing at room temperature for 12 hours, heat the mixture to 75°C and maintain this temperature for another 4 hours; collect the orange product by centrifugation and thoroughly wash it with anhydrous methanol to remove the unloaded pentacarbonyl manganese bromide; finally, place the product biligand metal-organic framework@pentacarbonyl manganese bromide in the dark for further use; Step S3: Dissolve CuCl2·2H2O and sodium citrate dihydrate in 50 ml of aqueous solution by ultrasonication; then, add 50 μL of sodium sulfide solution at room temperature with stirring for 5 minutes; then, heat the reaction solution at 90°C for 30 minutes until a dark green solution is obtained; transfer the mixture into ice water; obtain CuS and store at 4°C; Step S4: For the biligand metal-organic framework @ pentacarbonyl manganese bromide @ copper sulfide, the prepared CuS aqueous solution was mixed with the biligand metal-organic framework @ pentacarbonyl manganese bromide and stirred for 1 h, then transferred to 90°C in an oil bath and stirred for 4 h; the product biligand metal-organic framework @ pentacarbonyl manganese bromide @ copper sulfide was collected by centrifugation and washed with deionized water; 60 μL of 2 mg / mL biligand metal-organic framework @ pentacarbonyl manganese bromide @ copper sulfide was resuspended in 2 mM PBS to obtain the drug.

2. The method for preparing a nanomedicine for multi-modal synergistic tumor therapy according to claim 1, characterized in that: In step S1, the N,N-dimethylformamide is 1-5 ml, tetrakis(4-carboxyphenyl)porphine ferric chloride is 0.1-1 mg, 2,2'-bipyridine-5,5'-dicarboxylic acid is 20-40 mg, ZrOCl2·8H2O is 10-30 mg, and benzoic acid is 50-300 mg.

3. The method for preparing a nanomedicine for multi-modal synergistic tumor therapy according to claim 1, characterized in that: In step S2, the amount of the biligand metal organic framework is 10-40 mg, and the amount of manganese bromide pentacarbonyl is 10-50 mg.

4. The method for preparing a nanomedicine for multi-modal synergistic tumor treatment according to claim 1, characterized in that: In step S3, the amount of CuCl2·2H2O is 5-20 mg, the amount of sodium citrate dihydrate is 5-20 mg, and the amount of sodium sulfide solution is 100-300 mg / mL.

5. The method for preparing a nanomedicine for multi-modal synergistic tumor treatment according to claim 1, characterized in that: In step S4, the CuS aqueous solution is 3-10 ml, and the biligand metal organic framework@pentacarbonyl manganese bromide is 1-5 mg.

6. A nanomedicine for multi-modal synergistic tumor treatment, prepared by the preparation method according to any one of claims 1 to 5.

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