A biomimetic nanoscale bimetallic organic framework loaded plasmid material, a preparation method and application thereof

Abstract

The application discloses a kind of bionic nanoscale bimetallic organic framework loaded plasmid materials and preparation method and application thereof, belong to nanometer material and tumor drug technical field.The material prepared by the application uses bionic nanoscale bimetallic organic framework as nanocarrier, successfully loads plasmid gene drug, and is coated with tumor cell homologous cell membrane on its surface, realizes the efficient targeted delivery of iron, copper and gene drug, realizes the purpose of tumor cell iron / copper death and Fenton / Fenton-like reaction synergistic treatment drug-resistant tumor, simultaneously provides new thought and direction for developing series multifunctional nanodiagnosis and treatment platform.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials and tumor drug technology, specifically relating to a biomimetic nanoscale bimetallic organic framework supported plasmid material, its preparation method and application. Background Technology

[0002] Tumor drug resistance limits the effectiveness of apoptosis-based drug therapies and is a major cause of chemotherapy failure. Therefore, utilizing other forms of non-apoptotic cell death to eliminate drug-resistant tumor cells is of great significance. Ferroplasmosis and copper pluvialis are unique metal ion-dependent cell death mechanisms that differ from other forms of cell death, such as apoptosis, at the morphological, biological, and genetic levels, and possess certain potential for treating drug-resistant tumors. Furthermore, iron and copper overload within tumor cells can trigger Fenton and Fenton-like reactions in acidic environments, producing excessive cytotoxic reactive oxygen species, leading to tumor cell death.

[0003] Loading iron and copper ions onto nanomaterials can induce ferroptosis and copper death in tumor cells. However, existing nanomaterials have the following problems: metal ions are difficult to effectively fix onto the carrier. Once free ions are formed in the body, they will produce metal toxicity and be rapidly metabolized and excreted from the body, thus failing to exert a therapeutic effect. Even if metal ions are fixed onto nanomaterials, it is difficult to target tumor cells in the systemic circulation, which may lead to the killing of normal cells while killing tumor cells.

[0004] In the field of cancer treatment, gene therapy can efficiently and specifically regulate the expression of target genes in tumor cells, thereby achieving precision cancer treatment. Therefore, selective gene delivery can enable tumor gene therapy targeting the mechanisms of ferroptosis and copper death. However, exogenous genes require specific delivery vehicles to enter cells and exert their effects, and are highly sensitive to various enzymes in the body, making them easily degraded and eliminated. Therefore, selecting suitable gene drug delivery vectors to maintain the activity of the gene drugs and achieve effective targeted release is crucial for the success of gene therapy and remains one of the challenges in tumor gene therapy research. Summary of the Invention

[0005] To address the challenges in existing technologies, this invention provides a biomimetic nanoscale bimetallic organic framework-loaded plasmid material, its preparation method, and its application. By preparing a dual-ion (iron / copper) metal-organic framework nanocarrier, plasmid gene drugs are loaded onto this carrier, and tumor cell homologous cell membranes are coated on its surface. This achieves efficient targeted delivery of iron, copper, and gene drugs, and realizes the synergistic treatment of drug-resistant tumors through tumor cell iron / copper death and Fenton / Fenton-like response.

[0006] In a first aspect, the present invention provides a method for preparing biomimetic nanoscale bimetallic organic framework supported plasmid materials, comprising the following steps:

[0007] Disulfide-modified phloroglucinol was synthesized from phloroglucinol and 3,3'-dithiodipropionic anhydride.

[0008] The nanocarrier was prepared by dissolving the disulfide-modified phloroglucinol, iron source, and copper source together, followed by ultrasonication and heat treatment.

[0009] The nanocarrier was loaded with plasmids, and then cell membrane fragments were added. The mixture was then sonicated and centrifuged to obtain a biomimetic nanoscale bimetallic organic framework-loaded plasmid material with homologous cell membrane modification.

[0010] Preferably, the 3,3'-dithiodipropionic anhydride is prepared by the following method:

[0011] Dissolve 3,3'-dithiodipropionic acid in acetyl chloride and reflux at 60-70°C. Cool the refluxed solution to room temperature and then evaporate the solvent.

[0012] It is obtained by repeated washing and drying.

[0013] Preferably, the disulfide-modified phloroglucinol is synthesized by the following method:

[0014] The resorcinol, 3,3'-dithiodipropionic anhydride, dichloromethane (EDC), and 4-dimethylaminopyridine (DMAP) are dissolved in an organic solvent, heated to 45–60 °C and stirred in the dark for 60–80 h, and the solvent and unreacted raw materials are removed to obtain the final product.

[0015] Preferably, the molar ratio of phloroglucinol, 3,3'-dithiodipropionic anhydride, dichloromethane, and 4-dimethylaminopyridine is 1-2:5-10:10-15:10-15.

[0016] Preferably, the iron source is FeCl3·6H2O, and the copper source is CuCl2·2H2O;

[0017] The molar ratio of the disulfide-modified phloroglucinol, iron source, and copper source is 6:1 to 3:1 to 3.

[0018] Preferably, the ultrasonic treatment time is not less than 60 minutes, the heat treatment temperature is 110-140°C, and the heat treatment time is 40-50 hours.

[0019] Preferably, after heat treatment, the reaction mixture is centrifuged and repeatedly washed, wherein the centrifugation speed is 7000-9000 rpm and the centrifugation time is 12-18 min.

[0020] Preferably, the plasmid comprises FDX1 plasmid and p53 plasmid in a mass ratio of 1:1, and the concentration of the plasmid is 200-300 ng / μL;

[0021] The mass ratio of the plasmid to the nanocarrier is 1 to 2:500.

[0022] Preferably, the cell membrane fragment is prepared by the following method: collecting breast cancer 4T1 cells, washing them clean, centrifuging them at 3-5°C, suspending them, and using liquid nitrogen to freeze-thaw 6 times to destroy the cell membrane;

[0023] Then, after continuous centrifugation of the solution, the supernatant was collected to obtain the membrane fragment solution;

[0024] The supernatant was continuously centrifuged and the precipitated cell membrane fragments were collected.

[0025] The cell membrane fragments were washed with RNase-free water and resuspended to obtain the final product.

[0026] Preferably, the cell membrane fragment is a tumor-derived cell membrane (CM) fragment.

[0027] In a second aspect, the present invention provides a biomimetic nanoscale bimetallic organic framework-supported plasmid material prepared by any of the preparation methods described in the first aspect.

[0028] Thirdly, the present invention provides the application of the biomimetic nanoscale bimetallic organic framework-supported plasmid material described in the second aspect in the preparation of tumor gene therapy drugs.

[0029] In this invention, the prepared biomimetic nanoscale bimetallic organic framework loaded plasmid material can be used to produce gene therapy drugs for drug-resistant tumors, achieving the goal of synergistic treatment of drug-resistant tumors through iron / copper death of tumor cells and Fenton / Fenton-like reaction.

[0030] The technical solutions provided in this application have at least the following advantages compared with the prior art:

[0031] 1. This invention discloses a method for preparing biomimetic nanoscale bimetallic organic framework-loaded plasmid materials. By coating the surface of the metal-organic framework with tumor cell homologous cell membranes, the stability and biocompatibility of the nanocarrier are increased, protecting the loaded plasmids from degradation by various enzymes, and enabling the nanomaterials to effectively target tumor cells. Furthermore, it can serve as a gene therapy for drug-resistant tumors, fully and effectively utilizing the iron / copper ion-dependent characteristics of ferroptosis and copper death, as well as the Fenton and Fenton-like reactions of iron / copper ions in the acidic environment of tumor cells. These two factors synergistically kill drug-resistant tumor cells.

[0032] 2. The biomimetic nanoscale bimetallic organic framework-loaded plasmid material prepared in this invention is loaded with plasmids that induce ferroptosis and copper apoptosis, achieving efficient delivery of iron, copper, and gene drugs. Furthermore, the iron / copper biionic metal-organic framework has a large specific surface area and numerous hydrophobic sites, facilitating cell membrane adsorption. Cell membrane encapsulation can be achieved through sonication, effectively mimicking adherent cells, improving the biocompatibility and stability of the nanomaterial, and making it easier for tumor cells to recognize and take up. The establishment of this nanoplatform enables the synergistic effect of iron / copper overload and gene drugs to induce ferroptosis and copper apoptosis, and also achieves Fenton and Fenton-like reactions in an acidic environment of iron / copper ions. In addition, this research strategy can be extended to the encapsulation of easily inactivated gene drugs such as siRNA and miRNA, as well as other types of metal-organic framework materials, to develop a series of multifunctional nanotherapeutic platforms. Attached Figure Description

[0033] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0034] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 Transmission electron microscopy images of CM-Plas@MOFs and Plas@MOFs;

[0036] Figure 2 The particle size of CM-Plas@MOFs and Plas@MOFs;

[0037] Figure 3 A schematic diagram showing the change in the content of residual membrane proteins in the reaction solution after MOFs coat homologous cell membranes;

[0038] Figure 4 A schematic diagram comparing the stability of CM-Plas@MOFs and Plas@MOFs;

[0039] Figure 5 Transmission electron microscopy images and elemental analysis diagrams of Fe-MOFs, Cu-MOFs, Fe / Cu-MOFs and CM@MOFs;

[0040] Figure 6 A schematic diagram comparing the targeting capabilities of CM-Plas@MOFs and Plas@MOFs;

[0041] Figure 7This is a schematic diagram comparing the cell lethality of CM-Plas@MOFs and Plas@MOFs. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0043] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0044] The principles and features of the present invention are described below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, specific conditions or manufacturer-recommended conditions should be followed in the embodiments. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0045] Example 1

[0046] This embodiment discloses a biomimetic nanoscale bimetallic organic framework supported plasmid material, which is prepared by the following method:

[0047] Step S1: Preparation of 3,3'-dithiodipropionic anhydride

[0048] 2 g of 3,3'-dithiodipropionic acid was added to 10 mL of acetyl chloride and refluxed at 65 °C for 2 h. The resulting solution was cooled to room temperature and the solvent was evaporated. The precipitate was then repeatedly washed with diethyl ether, and the resulting product, 3,3'-dithiodipropionic anhydride, was dried overnight in a vacuum desiccator.

[0049] Step S2: Synthesis of disulfide-modified phloroglucinol

[0050] Phloroglucinol (1.5 mmol), 3,3'-dithiodipropionic anhydride (6 mmol), EDC (12 mmol) and DMAP (12 mmol) were dissolved in 150 mL of tetrahydrofuran and heated to 50 °C and stirred in the dark for 72 h.

[0051] Then, the solvent is removed by a rotary evaporator;

[0052] The reaction mixture was repeatedly washed with methanol and ice-cold diethyl ether to remove unreacted phloroglucinol and 3,3'-dithiodipropionic anhydride until the supernatant was colorless.

[0053] Step S3: Preparation of Fe / Cu-MOFs

[0054] Disulfide-modified phloroglucinol (6 mmol), FeCl3·6H2O (2 mmol) and CuCl2·2H2O (2 mmol) were dissolved in 30 mL of DMF, and the mixture was ultrasonically treated in a sealed autoclave for 60 min, and then heated to 120 °C for 48 h.

[0055] The reaction mixture was then centrifuged at 8000 rpm for 15 min and washed repeatedly with methanol and DMF until the supernatant was colorless to remove unreacted molecules and obtain Fe / Cu-MOFs.

[0056] Step S4: Loading FDX1 and p53 plasmids

[0057] Add 5 μg FDX1 plasmid (250 ng / μL) and 5 μg p53 plasmid (250 ng / μL) to 10 mL of Fe / Cu-MOFs (0.5 mg / mL) prepared in step S3, stir in the dark for 12 h, and centrifuge and wash to obtain Plas@MOFs.

[0058] Step S5: Preparation of tumor-derived cell membrane (CM) fragments

[0059] Collect 3×10 7 4T1 breast cancer cells were washed three times with 1×PBS, centrifuged at 4°C (2000 rpm) for 5 min, then suspended in 0.1×PBS and subjected to 6 freeze-thaw cycles with liquid nitrogen to disrupt the cell membrane.

[0060] Then the solution was centrifuged at 4°C (3000 rpm) for 10 min, and the supernatant was collected to obtain the membrane fragment solution;

[0061] The collected supernatant was centrifuged at 4°C (14000 rpm) for 10 min to collect precipitated cell membrane fragments. The precipitated fragments were washed three times with RNase-free water. Finally, the cell membrane fragments were resuspended in RNase-free water and stored at -20°C.

[0062] Step S6: Modify Plas@MOFs with homologous cell membranes (CM)

[0063] Add 25 μL of cell membrane fragment to 250 μL of Plas@MOFs (FDX1 / p53@Fe / Cu-MOFs) prepared in step S5, sonicate for 1 h, centrifuge at 4 °C (10000 rpm) for 10 min, and wash 3 times to obtain CM-Plas@MOFs.

[0064] Comparative Example 1

[0065] The difference between this comparative example and Example 1 is that in step S3, only FeCl3·6H2O is used to react with disulfide-modified phloroglucinol to prepare Fe-MOFs.

[0066] Comparative Example 2

[0067] The difference between this comparative example and Example 1 is that in step S3, only CuCl3·2H2O is used to react with disulfide-modified phloroglucinol to prepare Cu-MOFs.

[0068] Comparative Example 3

[0069] The difference between this comparative example and Example 1 is that plasmid loading is not performed, that is, step S4 is omitted, and the Fe / Cu-MOFs of step S3 are directly modified with the homologous cell membrane (CM) of step S5 to obtain CM@MOFs.

[0070] Among them, such as Figure 1 As shown, Figure 1 The left image shows a transmission electron microscope (TEM) image of Plas@MOFs loaded with FDX1 and p53 plasmids in Example 1, and the right image shows a TEM image of CM-Plas@MOFs obtained by modifying Plas@MOFs with homologous cell membranes (CM) in Example 1. It can be seen that both Plas@MOFs and CM-Plas@MOFs exhibit good dispersibility and uniform particle size.

[0071] like Figure 2 As shown, Figure 2 The particle sizes of Plas@MOFs and CM-Plas@MOFs are shown. It can be seen that the particle size of CM-Plas@MOFs increased from 185nm to 214nm, which means the particle size increased. This indicates that the surface of the dual-ion (iron and copper) metal-organic framework (MOF) nanocarrier of the present invention was successfully coated with cell membrane.

[0072] like Figure 3 As shown, Figure 3 The change in the amount of remaining membrane proteins in the reaction solution after MOFs were coated with homologous cell membranes shows that it decreased significantly, indicating that the cell membrane was successfully coated on the surface of MOFs.

[0073] like Figure 4 As shown, Figure 4The purpose was to test the stability of Plas@MOFs and CM-Plas@MOFs. It can be seen that CM-Plas@MOFs with cell membranes are stable in DMEM culture medium, and the plasmid release is about 5% after long-term incubation; Plas@MOFs without cell membranes are unstable in DMEM culture medium, and the plasmid release reaches about 95% after 2 hours.

[0074] like Figure 5 As shown, Figure 5 Transmission electron microscopy (TEM) images and elemental analysis diagrams of Fe-MOFs (Comparative Example 1), Cu-MOFs (Comparative Example 2), Fe / Cu-MOFs, and CM@MOFs (Comparative Example 3) are shown. TEM images reveal that the cell membrane thickness on the surface of CM@MOFs is approximately 12 nm. Elemental analysis shows that the nanoparticles contain Fe, Cu, and P, indicating successful loading of Fe and Cu and successful encapsulation of the cell membrane.

[0075] Since both cell membranes and plasmids contain phosphorus (P), the presence of P in the empty CM@MOFs vector proves that the MOFs successfully coated the cell membrane; however, if P is detected in CM-Plas@MOFs, it cannot be determined whether the P originated from the cell membrane or the plasmid. Therefore, from... Figure 5 As can be seen, the method of the present invention can prove that MOFs successfully coat the cell membrane.

[0076] like Figure 6 As shown, Figure 6 To verify the targeting ability of CM-Plas@MOFs, the nanomaterials were co-incubated with 4T1 cells, and the fluorescence intensity of the nanomaterials entering the cells was detected by flow cytometry. The fluorescence intensity of CM-Plas@MOFs coated with cell membranes (99.5%) was significantly higher than that of Plas@MOFs without cell membranes (28.8%), indicating that coating with homologous cell membranes can improve the targeting ability of nanomaterials.

[0077] like Figure 7 As shown, Figure 7 To verify the cell lethality of CM-Plas@MOFs, the nanomaterials were co-incubated with 4T1 cells, and the lethality was detected by flow cytometry. The cell lethality rate (proportion of necrotic or unhealthy cells) of CM-Plas@MOFs was approximately 39.5%, while that of Plas@MOFs was approximately 13.86%.

[0078] Various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Furthermore, whenever a numerical range is referred to herein, it means including any referenced number (fraction or integer) within the referred range.

[0079] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for preparing biomimetic nanoscale bimetallic organic framework supported plasmid materials, characterized in that, Includes the following steps: Disulfide-modified phloroglucinol was synthesized from phloroglucinol and 3,3'-dithiodipropionic anhydride. The nanocarrier was prepared by dissolving the disulfide-modified phloroglucinol, iron source, and copper source together, followed by ultrasonication and heat treatment. The nanocarrier was loaded with plasmids, and then cell membrane fragments were added. The mixture was then sonicated and centrifuged to obtain a biomimetic nanoscale bimetallic organic framework-loaded plasmid material with homologous cell membrane modification. The plasmids include FDX1 plasmid and p53 plasmid in a mass ratio of 1:1, and the concentration of the plasmids is 200~300 ng / μL; The mass ratio of the plasmid to the nanocarrier is 1~2:500; The cell membrane fragment is a tumor-derived cell membrane fragment.

2. The method for preparing biomimetic nanoscale bimetallic organic framework supported plasmid material according to claim 1, characterized in that, The 3,3'-dithiodipropionic anhydride was prepared by the following method: Dissolve 3,3'-dithiodipropionic acid in acetyl chloride and reflux at 60-70°C. Cool the refluxed solution to room temperature and then evaporate the solvent. It is obtained by repeated washing and drying.

3. The method for preparing biomimetic nanoscale bimetallic organic framework supported plasmid material according to claim 1, characterized in that, The disulfide-modified phloroglucinol was synthesized by the following method: The resorcinol, 3,3'-dithiodipropionic anhydride, EDC, and 4-dimethylaminopyridine are co-dissolved in an organic solvent, heated to 45-60°C and stirred in the dark for 60-80 hours. The solvent and unreacted raw materials are then removed to obtain the final product.

4. The method for preparing biomimetic nanoscale bimetallic organic framework supported plasmid material according to claim 3, characterized in that, The molar ratio of phloroglucinol, 3,3'-dithiodipropionic anhydride, EDC, and 4-dimethylaminopyridine is 1~2:5~10:10~15:10~15.

5. The method for preparing biomimetic nanoscale bimetallic organic framework supported plasmid material according to claim 1, characterized in that, The iron source is FeCl3·6H2O, and the copper source is CuCl2·2H2O; The molar ratio of the disulfide-modified phloroglucinol, iron source, and copper source is 6:1~3:1~3.

6. The method for preparing biomimetic nanoscale bimetallic organic framework supported plasmid material according to claim 1 or 5, characterized in that, The ultrasonic treatment time is not less than 60 minutes, the heat treatment temperature is 110~140℃, and the heat treatment time is 40~50 hours.

7. The method for preparing biomimetic nanoscale bimetallic organic framework supported plasmid material according to claim 6, characterized in that, After heat treatment, the reaction mixture is centrifuged and repeatedly washed, with the centrifugation speed being 7000~9000 rpm and the centrifugation time being 12~18 min.

8. The biomimetic nanoscale bimetallic organic framework supported plasmid material prepared by the preparation method of the biomimetic nanoscale bimetallic organic framework supported plasmid material according to any one of claims 1 to 7.

9. The application of the biomimetic nanoscale bimetallic organic framework-supported plasmid material according to claim 8 in the preparation of tumor gene therapy drugs.

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