MnZn-LDH-3MA layered double hydroxide material system as well as preparation method and application thereof

Abstract

The invention relates to the technical field of layered double hydroxide materials, in particular to a MnZn-LDH-3MA layered double hydroxide material system and a preparation method and application thereof.The preparation method comprises the steps that a layered double hydroxide MnZn-LDH precursor is prepared in an alkaline environment through a hydrothermal method, then 3-methyladenine 3MA is loaded on the precursor, and the final sample MnZn-LDH-3MA is prepared. The MnZn-LDH-3MA prepared by the invention can be effectively accumulated in a tumor, excessive H < + > in a tumor microenvironment is effectively neutralized, and the local sonodynamic therapy of the tumor is effectively realized under ultrasonic irradiation. The preparation method has the advantages of low cost of required raw materials, simplicity in operation, short time consumption, high drug loading capacity, uniform particle size and the like, and tumor growth can be effectively inhibited through autophagy inhibition generated by active oxygen in tumor treatment.

Description

Technical Field

[0001] The invention relates to the technical field of layered double hydroxide materials, and in particular to a MnZn-LDH-3MA layered double hydroxide material system and a preparation method and application thereof. Background Art

[0002] As the burden of cancer continues to grow worldwide, various diagnostic and treatment methods for cancer are also being developed. Although chemotherapy, radiotherapy and other traditional methods of treating cancer have certain efficacy, they often have disadvantages such as poor tumor targeting and large toxic side effects. In order to overcome this problem, nanomaterial medicine strives to develop anti-cancer materials with good targeting, high treatment efficiency and good stability. Sonodynamic therapy (SDT) is a non-invasive, precise treatment range, ultrasound responsiveness and low side effects cancer treatment method. Sonosensitizers and low-frequency ultrasound can act on tumors at the same time. Generally speaking, sonosensitizers with good tumor targeting are selected, and when they accumulate at the tumor site, ultrasound is used to activate the sonosensitizers, and cytotoxic reactive oxygen species (ROS) are efficiently generated to achieve non-invasive tumor treatment. In addition, ultrasound can not only be used to activate sonosensitizers, but also because of its high penetration into biological tissues, it is often used for disease diagnosis and tumor imaging in clinical treatment.

[0003] Metal drugs have been shown to be a potential class of anti-tumor chemotherapy drugs in research. Manganese is a nutrient inorganic trace element that participates in a variety of physiological processes. Due to its good redox properties, Mn can decompose H2O2 through a Fenton-like reaction to produce a large number of oxidation products, which can directly kill tumors. In addition, Mn 2+ As an immune adjuvant, it can also activate the interferon gene stimulator (STING) signaling pathway to produce an immune response. Zinc ions are cofactors of many enzymes and transcription factors, and as structural ions, they help stabilize the enzyme structure. Studies have shown that excessive Zn 2+ It has anti-tumor effects in many tumors. 2+ The anti-tumor mechanism of nanomedicine mainly includes Zn 2+ Overload-mediated mitochondrial dysfunction and subsequent antitumor immune responses. 2+ It inhibits mitochondrial oxidative respiration, leading to mitochondrial dysfunction, thereby reducing intracellular ATP levels.

[0004] Autophagy refers to the process of using lysosomes to degrade damaged organelles and certain macromolecules under the mediation of autophagy-related genes. It plays a unique role in maintaining the homeostasis of the intracellular environment and belongs to programmed cell death. The basic process of autophagy can be divided into five steps: 1. Initiation of autophagy-inducing signals, 2. Formation of isolated phagosomes, 3. Extension and closure of the double-layer membrane structure that wraps the cargo to form autophagosomes, 4. Docking and fusion of autophagosomes and lysosomes, and 5. Lysosomal enzymes degrade the contents of autophagic vacuoles. Autophagy plays a "double-edged sword" role in the occurrence and treatment of tumors. Excessive tumor autophagy will clear damaged mitochondria in cells, prevent the accumulation of ROS, and decompose them into nucleotides necessary for DNA repair through metabolism.

[0005] Therefore, nanomedicine researchers are trying to combine multiple drugs to achieve combined treatment of tumors. Using the advantages of different methods to jointly create better treatment ideas, combining oxidative stress with autophagy inhibition to jointly inhibit tumor growth is an innovative, feasible and effective treatment method. Summary of the invention

[0006] The purpose of the present invention is to solve the shortcomings of the prior art and propose a MnZn-LDH-3MA layered double hydroxide material system and its preparation method and application. The prepared MnZn-LDH-3MA can effectively inhibit tumor autophagy, damage tumor mitochondria, and achieve the effect of killing tumors.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A MnZn-LDH-3MA layered double hydroxide material includes a manganese-zinc layered double hydroxide loaded with 3-methyladenine (3MA). A layered double hydroxide (MnZn-LDH) precursor is first prepared by a hydrothermal method in an alkaline environment, and then 3MA is loaded on the precursor to prepare a final sample MnZn-LDH-3MA.

[0009] The present invention also provides a method for preparing a MnZn-LDH-3MA layered double hydroxide material, comprising the following steps:

[0010] Step 1, dissolving Mn(NO3)2·4H2O in pure water to prepare a manganese metal salt solution;

[0011] Step 2, dissolving Zn(NO3)2·6H2O in pure water to prepare a zinc metal salt solution;

[0012] Step 3, dissolving NaOH in pure water;

[0013] Step 4, mixing the manganese metal salt solution prepared in step 1 and the zinc metal salt solution prepared in step 2, then pouring the mixed liquid into the aqueous solution of NaOH, and vigorously stirring at room temperature for 30 minutes;

[0014] Step 5, transferring the reaction product into an autoclave, reacting at 100°C for 12h;

[0015] Step 6, after the autoclave is cooled to room temperature, the precipitate is collected and purified by centrifugation, and dried overnight under vacuum conditions to obtain MnZn-LDH;

[0016] Step 7: Mix the MnZn-LDH prepared in step 6 with 3MA to prepare a MnZn-LDH-3MA material.

[0017] Preferably, in step 1 and step 2, the molar ratio of Mn(NO3)2·4H2O to Zn(NO3)2·6H2O is 2:1.

[0018] Preferably, in step 6, the solvent used for purification is pure water.

[0019] Preferably, in step 6, the MnZn-LDH is lamellar nanoparticles with uniform particle size and uniform morphology.

[0020] The present invention also provides an application of the MnZn-LDH-3MA layered double hydroxide material prepared by the above preparation method in the preparation of anti-tumor drugs for treatment.

[0021] The present invention also provides an application of the MnZn-LDH-3MA layered double hydroxide material prepared by the above preparation method in the preparation of sonodynamic drugs, specifically: through the effective accumulation of LDH in the tumor, sonodynamic therapy of the tumor site is achieved under ultrasonic irradiation.

[0022] The present invention also provides an application of the MnZn-LDH-3MA layered double hydroxide material prepared by the above preparation method in preparing a drug carrier preparation.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The MnZn-LDH obtained by the present invention has uniform size and good dispersibility, and the synthesis method is simple and fast, which is convenient for production and is beneficial to the penetration and long-term retention of solid tumors.

[0025] 2. The MnZn-LDH obtained in the present invention has good biosafety and high stability, can generate a large amount of active oxygen under ultrasonic irradiation, and can be used for SDT treatment of tumors.

[0026] 3. The MnZn-LDH-3MA obtained by the present invention can effectively inhibit tumor autophagy, damage tumor mitochondria, and achieve the effect of killing tumors.

[0027] 4. The MnZn-LDH-3MA obtained by the present invention can effectively accumulate in tumors, and LDH can effectively neutralize excessive H in the tumor microenvironment. + , under ultrasound irradiation, local sonodynamic therapy of tumors can be effectively achieved.

[0028] 5. The raw materials required by the present invention have many advantages such as low cost, simple operation, short time consumption, and uniform particle size. In tumor treatment, they can effectively inhibit tumor growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a particle size distribution diagram of MnZn-LDH-3MA prepared in Example 1 of the present invention;

[0030] Figure 2 This is a transmission electron microscope image of MnZn-LDH-3MA prepared in Example 1 of the present invention;

[0031] Figure 3 This is a sonodynamic performance diagram of the MnZn-LDH prepared in Example 1 of the present invention;

[0032] Figure 4 This is a graph showing the acid neutralization ability of MnZn-LDH prepared in Example 1 of the present invention at the cellular level;

[0033] Figure 5 This is a diagram for evaluating the toxicity of MnZn-LDH-3MA prepared in Example 1 of the present invention to 4T1 cells under ultrasonic conditions;

[0034] Figure 6 This is a graph showing the ability of MnZn-LDH-3MA prepared in Example 1 of the present invention to generate reactive oxygen species (ROS) in cells under ultrasonic conditions;

[0035] Figure 7 This is a tumor treatment curve diagram of the MnZn-LDH-3MA prepared in Example 1 of the present invention on tumor-bearing mice. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, thereby making a clearer definition of the protection scope of the present invention. The embodiments described in the present invention are only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention.

[0037] Embodiment 1:

[0038] A MnZn-LDH-3MA layered double hydroxide material comprises the following raw materials:

[0039] Manganese nitrate tetrahydrate, zinc nitrate hexahydrate, sodium hydroxide;

[0040] Manganese nitrate tetrahydrate and zinc nitrate hexahydrate are prepared in their respective aqueous solutions.

[0041] The preparation method of the MnZn-LDH-3MA layered double hydroxide material comprises the following steps:

[0042] S1. Dissolve Zn(NO3)2·6H2O in pure water to prepare a zinc metal salt solution;

[0043] S2. Dissolve Mn(NO3)2·4H2O in pure water to prepare a manganese metal salt solution;

[0044] S3, dissolving NaOH in pure water to prepare a NaOH aqueous solution;

[0045] S4, mix the prepared zinc metal salt solution and manganese metal salt solution to make the molar ratio of Mn(NO3)2·4H2O to Zn(NO3)2·6H2O 2:1, pour into the NaOH aqueous solution, and stir vigorously at room temperature for 30 minutes;

[0046] S5, transferring the reaction product to an autoclave, reacting at 100°C for 12h;

[0047] S6. After the autoclave is cooled to room temperature, the precipitate is collected and purified by centrifugation, and dried overnight under vacuum conditions to obtain MnZn-LDH;

[0048] S7. Mix the prepared MnZn-LDH with 3MA to prepare a MnZn-LDH-3MA material.

[0049] 5 μg / mL MnZn-LDH nanoparticles and 1 mg / mL 1,3-diphenylisobenzofuran (DPBF) were dissolved in 3.0 mL ultrapure water and ultrasonicated (ultrasonic frequency 1 MHz, duty cycle 80%, power 1.0 W / cm 2 ) At different times, the ultraviolet absorption of the solution was tested. DPBF is a probe for detecting active oxygen. With the generation of active oxygen, the absorption of its characteristic peak at 410 nm will gradually decrease.

[0050] The sonodynamic performance of the above-mentioned sonogenic nanoparticles MnZn-LDH in vitro is as follows Figure 3As shown; with the extension of ultrasound time, it can be observed that the absorption value of the characteristic peak of DPBF at 410nm has gradually decreased, indicating that MnZn-LDH will produce a large amount of reactive oxygen under ultrasound irradiation conditions, and it has good sonodynamic performance in vitro.

[0051] The acid neutralization ability of the above-mentioned MnZn-LDH on 4T1 cells is as follows Figure 4 As shown, it can be observed that MnZn-LDH has a certain acid neutralization ability on 4T1 cells compared with the control group.

[0052] 40 μg / mL of MnZn-LDH was incubated with mouse breast cancer (4T1) cells, and the pH value of the cell supernatant of each group was detected every 12 hours. The experiment was terminated after 48 hours.

[0053] The above different concentrations of MnZn-LDH-3MA were incubated with mouse breast cancer (4T1) cells for 24 h, and then the cell activity was tested by standard MTT method. Ultrasound (ultrasound frequency 1 MHz, duty cycle 80%, power 1.0 W / cm 2 )5min, the toxicity to 4T1 cells was evaluated as follows Figure 5 As shown, it can be observed that under the action of ultrasound irradiation, MnZn-LDH-3MA has a certain toxicity to 4T1 cells.

[0054] Mouse breast cancer (4T1) cells were incubated with 40 μg / mL MnZn-LDH-3MA for 6 h and ultrasonicated (ultrasound frequency 1 MHz, duty cycle 80%, power 1.0 W / cm 2 ) for 5 minutes. DCFH-DA probe was added and its reactive oxygen species (ROS) were detected by laser confocal microscopy. DCFH-DA (dichlorodihydrofluorescein-acetoacetate) is a cell-permeable probe used to detect intracellular reactive oxygen species (ROS).

[0055] The ability of the sonosensitizing nanoparticles MnZn-LDH to generate reactive oxygen species (ROS) in cells is as follows: Figure 6 (Using DCFH-DA probe) as shown; DCFH-DA itself has no fluorescence, can freely pass through the cell membrane, is hydrolyzed by esterase to generate DCFH, accumulates in the cell, and is oxidized by reactive oxygen ROS to generate fluorescent DCF. Under 488nm excitation, the green fluorescence intensity is proportional to the level of reactive oxygen. Figure 6 As shown, the sonographically sensitive nanoparticles MnZn-LDH can produce a large amount of reactive oxygen species (ROS) under ultrasound and blue light irradiation conditions, indicating that it has good sonographic dynamic performance in cells.

[0056] The above-mentioned MnZn-LDH-3MA treatment curve for tumor-bearing mice is shown in Figure 7As shown; 4T1 tumor-bearing mice (tumor size: ~50mm 3 ) were randomly divided into 5 groups (6 in each group):

[0057] Group (1) was the control group (control), which was injected with PBS only;

[0058] Group (2) was the material group (3MA), which was injected with 3MA via the tail vein only;

[0059] Group (3) was the material group (MnZn-LDH), which was only injected with MnZn-LDH via the tail vein;

[0060] The fourth group was the material (LDH) ultrasound treatment group, where MnZn-LDH was injected into the tail vein + ultrasound conditions (ultrasound frequency 1 MHz, duty cycle 80%, power 1.0 W / cm 2 )5 minutes / piece;

[0061] The fifth group was the material (MnZn-LDH-3MA) ultrasound treatment group, where MnZn-LDH-3MA was injected into the tail vein + ultrasound conditions (ultrasound frequency 1 MHz, duty cycle 80%, power 1.0 W / cm 2 )5 minutes / piece.

[0062] 4 hours after the tail vein injection, the tumor was treated with ultrasound irradiation, and the above operation was repeated every three days for a total of three times. The tumor volume was measured and recorded every day. The tumor volume was determined by the following equation: Volume = length × width 2 / 2.

[0063] Compared with the control group, only the group (3) injected with MnZn-LDH via tail vein showed a certain anti-tumor effect, indicating that LDH can play a certain role; the group (4) injected with MnZn-LDH via tail vein + ultrasound irradiation showed a more obvious therapeutic effect than that of group (3), and the surface MnZn-LDH had a significant sonodynamic effect; the group (5) injected with MnZn-LDH-3MA via tail vein + ultrasound irradiation showed a significant therapeutic effect on tumor growth compared with other groups, indicating that this material can combine sonodynamics with inhibition of cell autophagy in tumor treatment to achieve a synergistic therapeutic effect.

[0064] In summary, the MnZn-LDH-3MA prepared in the present invention can effectively accumulate in tumors, and LDH can effectively neutralize excessive H in the tumor microenvironment. + , under ultrasound irradiation, local sonodynamic therapy of tumors can be effectively achieved.

[0065] The description and practice disclosed in the present invention are easy to think and understand for ordinary technicians in the technical field, and several improvements and modifications can be made without departing from the principles of the present invention. Therefore, modifications or improvements made without departing from the spirit of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A MnZn-LDH-3MA layered double hydroxide material, characterized in that: The method comprises a manganese-zinc layered double hydroxide loaded with 3-methyladenine 3MA. Firstly, a layered double hydroxide MnZn-LDH precursor is prepared in an alkaline environment by a hydrothermal method, and then 3MA is loaded on the precursor to prepare a final sample MnZn-LDH-3MA.

2. The method for preparing a MnZn-LDH-3MA layered double hydroxide material according to claim 1, characterized in that: The steps include: Step 1, dissolving Mn(NO3)2·4H2O in pure water to prepare a manganese metal salt solution; Step 2, dissolving Zn(NO3)2·6H2O in pure water to prepare a zinc metal salt solution; Step 3, dissolving NaOH in pure water; Step 4, mixing the manganese metal salt solution prepared in step 1 and the zinc metal salt solution prepared in step 2, then pouring the mixed liquid into the aqueous solution of NaOH, and vigorously stirring at room temperature for 30 minutes; Step 5, transferring the reaction product into an autoclave, reacting at 100°C for 12h; Step 6, after the autoclave is cooled to room temperature, the precipitate is collected and purified by centrifugation, and dried overnight under vacuum conditions to obtain MnZn-LDH; Step 7: Mix the MnZn-LDH prepared in step 6 with 3MA to prepare a MnZn-LDH-3MA material.

3. The method for preparing a MnZn-LDH-3MA layered double hydroxide material according to claim 2, characterized in that: In step 1 and step 2, the molar ratio of Mn(NO3)2·4H2O to Zn(NO3)2·6H2O is 2:

1.

4. The method for preparing a MnZn-LDH-3MA layered double hydroxide material according to claim 2, characterized in that: In step 6, the solvent used for purification is pure water.

5. The method for preparing a MnZn-LDH-3MA layered double hydroxide material according to claim 2, characterized in that: In step 6, MnZn-LDH is a lamellar nanoparticle with uniform particle size and uniform morphology.

6. Use of the MnZn-LDH-3MA layered double hydroxide material prepared by the preparation method according to any one of claims 2 to 5 in the preparation of anti-tumor drugs.

7. Use of the MnZn-LDH-3MA layered double hydroxide material prepared by the preparation method according to any one of claims 2 to 5 in the preparation of sonodynamic drugs.

8. Use of the MnZn-LDH-3MA layered double hydroxide material prepared by the preparation method according to any one of claims 2 to 5 in the preparation of a drug carrier preparation.

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