Preparation method and application of nano-drug for multi-mode synergistic tumor treatment

Abstract

The invention discloses a preparation method and application of a nano-drug for multi-mode synergistic tumor treatment, and relates to the technical field of biomedical materials, the technical key points are as follows: step S1, preparing a double-ligand metal organic framework by adopting a hydrothermal method; s2, dispersing the double-ligand metal organic framework in 10mL of absolute ethyl alcohol, adding pentacarbonyl manganese bromide (I), magnetically stirring, and thoroughly washing with absolute ethyl alcohol to remove unloaded pentacarbonyl manganese bromide (I); and finally obtaining the double-ligand metal organic framework (at) pentacarbonyl manganese bromide (I). S3, CuCl2. 2H2O and sodium citrate dihydrate are dissolved in 50 ml of an aqueous solution in an ultrasonic mode; and step S4, preparing the double-ligand metal organic framework (at) pentacarbonyl manganese bromide (I) (at) copper sulfide. The nano-drug provided by the invention is stable in structure, can be accurately released in a complex tumor microenvironment, can conveniently carry out multi-mode synergistic treatment on tumors, and can reduce side effects of each single treatment while obtaining a good treatment effect.

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 nano drug for multi-modal coordinated tumor treatment. Background Art

[0002] With the continuous development of tumor treatment technology, the limitations of single treatment methods are becoming increasingly apparent. Traditional chemotherapy and radiotherapy, while killing tumor cells, often cause inevitable damage to normal tissues, leading to serious side effects. In addition, due to the heterogeneity and complex microenvironment of tumors, single treatment methods are usually difficult to completely eliminate tumors, which can easily lead to drug resistance and recurrence. In order to overcome these challenges, multi-modality synergistic treatment strategies have gradually become a research hotspot in tumor treatment.

[0003] Nanomedicines have become ideal carriers for multi-modal synergistic therapy due to their unique physical and chemical properties, such as high surface area, good biocompatibility and controllable drug release characteristics. Nanomedicines can integrate multiple treatment methods such as chemotherapy, photothermal therapy, photodynamic therapy and immunotherapy on a single nanoplatform through targeted design to enhance the synergistic effect of treatment. At the same time, the drug loading capacity and controlled release mechanism of nanomedicines can help reduce damage to normal cells during treatment and improve the quality of life of patients.

[0004] However, how to efficiently prepare multifunctional nanomedicines and achieve their precise release and multi-modal synergistic effects in the complex tumor microenvironment is a technical problem that needs to be solved urgently. The present invention proposes an innovative nanomedicine preparation method, which aims to optimize the structure and function of nanocarriers, achieve multi-modal synergistic treatment of tumors, and thus improve the overall effect 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 a nano drug 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 nano drug 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 are ultrasonically dissolved in a round-bottom flask containing N,N-dimethylformamide; the reaction solution is stirred at 90°C for 3h; the oil bath is covered with aluminum foil to avoid light; after the reaction is completed, the nanoparticles are collected by centrifugation and washed three times with fresh N,N-dimethylformamide and anhydrous ethanol respectively; finally, the product is 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 bromide (I), and magnetic stirring; after uniformly mixing at room temperature for 12 hours, heating the mixture to 75°C and maintaining it at this temperature for another 4 hours; collecting the orange product by centrifugation, and washing it thoroughly with anhydrous methanol to remove the unloaded pentacarbonyl manganese bromide (I); finally, placing the product biligand metal organic framework @ pentacarbonyl manganese bromide (I) in the dark for further use;

[0010] Step S3, dissolving CuCl2·2H2O and sodium citrate dihydrate in 50 ml of aqueous solution by ultrasonication; then, adding 50 μL of sodium sulfide solution at room temperature for 5 min under stirring; then, heating the reaction solution at 90° C. for 30 min until a dark green solution is obtained; transferring the mixture into ice water; obtaining CuS and storing it at 4° C.;

[0011] Step S4, for the biligand metal organic framework @ pentacarbonyl manganese bromide (I) @ copper sulfide, the prepared CuS aqueous solution is mixed with the biligand metal organic framework @ pentacarbonyl manganese bromide (I) 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 bromide (I) @ copper sulfide is collected by centrifugation and washed with deionized water; 60 μL of 2 mg / mL biligand metal organic framework @ pentacarbonyl manganese bromide (I) @ 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 biligand metal organic framework is 10-40 mg, and pentacarbonyl manganese (I) bromide is 10-50 mg.

[0014] The present invention is further configured as follows: in step S3, the CuCl2·2H2O is 5-20 mg, the sodium citrate dihydrate is 5-20 mg, and the 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 bromide (I) is 1-5 mg.

[0016] A nano drug for multi-modal synergistic tumor treatment, the nano drug is composed of 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 prior art, the present solution has the following beneficial effects: the nanomedicine proposed in the present invention has a stable structure, can be accurately released in a complex tumor microenvironment, can conveniently perform multi-modal synergistic treatment on tumors, and can 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 the embodiment of the present invention;

[0020] Figure 2 The feasibility of multiple treatments of nanomedicine in solution in the embodiment of the present invention is verified, wherein (A) is a schematic diagram of multiple treatment effect evaluation based on DMCH in vitro, (B) is the fluorescence response of DMCH to singlet oxygen green fluorescent probe (Singlet Oxygen Sensor Green, SOSG) at different times under 660nm laser irradiation or without irradiation, (C) probe system (5μM CO probe + 20μM PdCl2 + 500μg / mL (D) is the UV-Vis absorption spectrum of 3,3',5,5'-tetramethylbenzidine (TMB) aqueous solution after different treatments. (E) is the UV-Vis absorption of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) for determining GSH level. (F) is the thermal image of PBS or DMCH aqueous solution at different times and laser power densities. (G) is the laser power density-dependent photothermal characteristics of DMCH (500 μg / mL). (H) is the laser power density-dependent photothermal characteristics of 500 μg / mL DMCH under 660 nm laser (0.86 W / cm 2) is the thermal cycle curve under irradiation, (I) is the photothermal and cooling curve of DMCH suspension (500 μg / mL), (J) is the curve of the negative natural logarithm of the cooling time and the temperature driving force, all the data prove its potential for multiple combined treatments in advance;

[0021] Figure 3 The nanomedicine in the present invention is used to treat tumors in nude mice. (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 ) is infrared thermal imaging at different times after irradiation, (C) is the imaging examination of five organs and tumors after 24 hours of treatment, (D) is the weight change of mice within 13 days of treatment, (E) is the time-dependent tumor growth curve of each treatment group, (F) is the average tumor weight of each group, (G) is the 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 solution of 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 described embodiments 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 creative work 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 in the embodiments of the present invention can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0024] Example:

[0025] (1) DMOF (double ligand 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 ultrasonically dissolved 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; 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 CuCl2·2H2O and 11.4 mg sodium citrate dihydrate were dissolved in 50 mL aqueous solution by ultrasound. Subsequently, 50 μL sodium sulfide solution (242 mg / mL) was added at room temperature with stirring for 5 min. Then, the reaction solution was 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, the prepared CuS aqueous solution (5 mL) with a concentration of 100 μg / mL was mixed with DMOF@MnCO (1 mg) and stirred for 1 h, then transferred to 90 °C in an oil bath and stirred for 4 h. The product 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 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 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 O2 oxidizes and 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 indicates that DMCH has a high efficiency in PDT. 1 O2 ability. In addition, MnCO, as a CO releaser, 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 determined by a standard TMB-based colorimetric method, 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 remained inactive. In the presence of 0.5 mg / mL DMCH and 1 mM H2O2, the TMB solution appeared 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 could not 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 It can be seen that when DMCH was added alone, the absorbance curve decreased slightly (black) compared with the solution with 10 mm GSH added alone (red), indicating that inactive DMCH could not significantly consume GSH. When H2O2 and GSH were added to 0.5 mg / 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, 808 nm laser was used at different power densities (0.30, 0.60, 0.86 and 1 W / 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 peak temperatures observed during five heating / cooling cycles at a power density of 1.34 W / m. Figure 2 H), indicating that DMCH maintained strong photothermal stability throughout the 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 analyzing the cooling curve ( Figure 2 J) and based on the equation in the experimental part, the photothermal conversion efficiency (η) of DMCH was 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. The therapeutic intervention procedures are detailed in 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 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, and the core temperature reached 37.9°C, which was significantly higher than 46.6°C after PBS treatment ( Figure 3 B). We studied the biodistribution of DMCH by intravenous injection of only Cy5-modified hairpin probes, and removed tumors and major organs for ex vivo fluorescence imaging. Cy5 fluorescence was the strongest in all tumor-bearing organs except the liver ( Figure 3 C), indicating that DMCH is efficiently accumulated in tumors. The liver efficiently metabolizes DMCH, which indicates reduced long-term toxicity to the organism. Body weight and tumor volume were measured daily during the treatment period. No significant changes in body weight were observed in all treatment groups ( Figure 3 D), indicating that the side effects of the nanoplatform are negligible. From the tumor volume curve, it can be seen that 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 modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for preparing a nano drug for multi-modal synergistic tumor treatment, characterized in that: The following steps are involved: 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 are ultrasonically dissolved in a round-bottom flask containing N,N-dimethylformamide; the reaction solution is stirred at 90°C for 3h; the oil bath is covered with aluminum foil to avoid light; after the reaction is completed, the nanoparticles are collected by centrifugation and washed three times with fresh N,N-dimethylformamide and anhydrous ethanol respectively; finally, the product is stored in the dark for further use; Step S2, dispersing the biligand metal organic framework in 10 mL of anhydrous ethanol, adding pentacarbonyl manganese bromide (I), and magnetic stirring; after uniformly mixing at room temperature for 12 hours, heating the mixture to 75°C and maintaining it at this temperature for another 4 hours; collecting the orange product by centrifugation, and washing it thoroughly with anhydrous methanol to remove the unloaded pentacarbonyl manganese bromide (I); finally, placing the product biligand metal organic framework @ pentacarbonyl manganese bromide (I) in the dark for further use; Step S3, dissolving CuCl2·2H2O and sodium citrate dihydrate in 50 ml of aqueous solution by ultrasonication; then, adding 50 μL of sodium sulfide solution at room temperature for 5 min under stirring; then, heating the reaction solution at 90° C. for 30 min until a dark green solution is obtained; transferring the mixture into ice water; obtaining CuS and storing it at 4° C.; Step S4, for the biligand metal organic framework @ pentacarbonyl manganese bromide (I) @ copper sulfide, the prepared CuS aqueous solution is mixed with the biligand metal organic framework @ pentacarbonyl manganese bromide (I) 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 bromide (I) @ copper sulfide is collected by centrifugation and washed with deionized water; 60 μL of 2 mg / mL biligand metal organic framework @ pentacarbonyl manganese bromide (I) @ copper sulfide is resuspended in 2 mM PBS to obtain the drug.

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

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

4. The method for preparing a nano drug 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 nano drug 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(I) bromide is 1-5 mg.

6. A nanomedicine for multi-modal synergistic tumor treatment, characterized by: The nano drug consists of 2,2'-bipyridine-5,5'-dicarboxylic acid, tetrakis(4-carboxyphenyl)porphine ferric chloride, pentacarbonyl manganese bromide and copper sulfide.

7. Use of a nano drug as described in claim 6 in multi-modality synergistic tumor therapy.

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